Methods and compositions to protect aquatic invertebrates from disease

ABSTRACT

Compositions and methods of protecting aquatic invertebrates from disease is shown. In one embodiment, dsRNA or antisense RNA to a nucleic acid molecule of the disease-causing microorganism is prepared and delivered to the animal. In another embodiment, a nucleic acid molecule of the disease-causing microorganism is delivered to the animal. In another embodiment, the RNA or nucleic acid molecule is delivered to the animal by replicon particle. In a further embodiment, the protective molecule is delivered to the digestive tract of the animal. Protection from disease is obtained.

RELATED APPLICATIONS

This application claims priority to previously filed and co-pendingprovisional application U.S. Ser. No. 61/407,297, filed Oct. 27, 2010;to previously filed and co-pending provisional application U.S. Ser. No.61/418,433, filed Dec. 1, 2010; to previously filed and co-pendingapplication U.S. Ser. No. 61/449,940 filed Mar. 7, 2011; to previouslyfiled and co-pending application U.S. Ser. No. 61/484,255 filed May 10,2011; to previously filed and co-pending application U.S. Ser. No.61/508,172 filed Jul. 15, 2011; and to previously filed and co-pendingapplication U.S. Ser. No. 61/525,332 filed Aug. 19, 2011, the contentsof each of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support awarded by The U.S.Department of Agriculture, National Institute of Food and AgricultureSBIR under contract 2010-33610-20936. The Government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 4, 2011, isnamed 150004US.txt and is 104,052 bytes in size.

BACKGROUND OF THE INVENTION

Aquaculture has become one of the fastest growing segments of foodanimal production in the world. This precipitous increase is beingdriven by decreasing stocks within wild fisheries and increasing demandfor seafood throughout the United States, Europe, and Japan. Disease inaquatic invertebrates is a serious problem for the industry.

For example, one of the fastest growing areas of demand is imported farmraised shrimp. According to FAO data, the last twenty years has seenfarmed aquatic and in particular farmed shrimp production of Litopenaeusvannamei, commonly the Pacific White Shrimp or the Pacific WhitelegShrimp, rise from 8000 metric tons in 1980 to 1,380,000 metric tonsproduced in 2004 Fisheries and Aquaculture Department.FishStatPlus—Fishery Statistical software 2009(www.fao.org/fishery/statistics/software/fishstat/en.) Most of thisdramatic increase can be accounted for by increases in productionthroughout Asia as they began to intensively culture Litopenaeusvannamei in lieu of the native Penaeus monodon, the black tiger shrimp,for export to the United States, specifically countries such as China,Thailand, Indonesia, and Vietnam. Fishery Statistical software 2009,supra. Following the introduction of Litopenaeus vannamei into Asia as adomesticated species, it became dominant, where in 2004 it accounted forhalf the shrimp produced globally. (Lightner, D. V., 2005. Biosecurityin shrimp farming: pathogen exclusion through use of SPF stock androutine surveillance. Journal of the World Aquaculture Society 36,229-248.)

As L. vannamei began to dominate cultivation abroad, so did domesticdemand in the United States. Shrimp imports rose from $1.6 billion to$3.7 billion from 1990-2004 (USDA Foreign Agriculture Service, 2005, USSeafood Imports Continue to Soar. International Trade Reports. Aug. 8,2005. Jul. 28, 2010) representing 34 percent of total seafood importsand 25 percent of total seafood consumption in 2004, respectively. As of2004, 70% of the United States seafood was imported with 40% of it beingfarm-raised, mostly cultured in southeast Asia. Rapidly increasingproduction of L. vannamei has outgrown demand, and led to pricedepression in international markets, mostly in the United States andEuropean Union. Farm value for 15-20 g size Pacific White shrimp hassteadily decreased from $5 US to about $3 in 2005. (Fisheries andAquaculture Department, 2009, supra).

As farm raised production continues to increase in market share incomparison to wild stocks, so does the impact of disease on shrimpfarming. Producers have adopted practices such as higher stockingdensities, smaller inland pond culture, and higher feeding rates toincrease competitiveness. This has led to an increasing vulnerability toinfectious disease, specifically viral pathogens followed by secondarybacterial infections. Viral diseases such as White Spot Syndrome Virus(WSSV) have become pandemic and resulted in worldwide losses in thebillions of dollars. For example, WSSV was first discovered in 1992after several outbreaks of a high mortality disease occurred in shrimpfarms in Taiwan (Chou, H.-Y., Huang, C.-Y., Wang, C.-H., Chiang, H.-C.,Lo, C.-F., 1995. Pathogenicity of a baculovirus infection causing whitespot syndrome in cultured penaeid shrimp in Taiwan. Diseases of AquaticOrganisms 23, 165-173.) It is estimated that Asia alone has lost over $6billion since 1992, and the Americas $1-2 billion since WSSV wasintroduced in 1999 (Lightner 2003 The penaeid shrimp viral pandemics dueto IHHNV, WSSV, TSV and YHV: history in the Americas and current status,Proceedings of the 32nd Joint UJNR Aquaculture Panel Symposium, Davisand Santa Barbara, Calif., USA, pp. 17-20.) In another example, Ecuadorexperienced dramatic losses, a 65% percent loss in production wasobserved after the introduction of WSSV and this accounted for, in lostexports alone, over a half billion US dollars. In addition, 130,000 jobswere lost and over 100,000 hectares of ponds were abandoned. (McClennen,C. White Spot Syndrome Virus, The Economic, Environmental and TechnicalImplications on the Development of Latin American Shrimp Farming. Masterof Arts in Law and Diplomacy Thesis. 2004.) Similarly, Peru experienceda precipitous drop in production to one tenth in 2000 of production in1998 with 85% of shrimp ponds being abandoned, and $9 million in lossesin feed costs alone. (McClennen, 2004, supra.) In China, it wasestimated that 80% of total production losses annually were attributedto WSSV. (Zhan, W.-B., Wang, Y.-H., 1998. White Spot Syndrome VirusInfection of Cultured Shrimp in China. Journal of Aquatic Animal Health10, 405-410.0

Currently there are no commercially available vaccines, therapeutics, orinterventions for these pathogens causing devastating economic losses toaquatic invertebrates and in particular in shrimp producing countries.

All references cited are incorporated herein by reference in theirentirety. Examples are provided by way of illustration and not intendedto limit the scope of the invention.

SUMMARY

Vaccines and compositions are described in which aquatic invertebratesare protection from adverse impact of a microorganism causing disease. Aprotective molecule, which may be a nucleic acid molecule of themicroorganism, a polypeptide encoded, an interfering RNA such as dsRNAor antisense RNA, or DNA encoding same or replicon vectors comprising orproducing any of the above are provided in a vaccine. Such moleculesprotect the animal from disease. Methods of administration andpreparation are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of VEE genome organization andreplication strategy and FIG. 1B is a schematic of the VEE repliconparticle vaccine and packaging system.

FIG. 2 is a diagram of the IMNV genome transcription and translationproducts showing regions targeted for RNAi, predicted protein productsare indicated by dark gray lines or gray shading, with target regionsfor dsRNA production indicated as thick black lines.

FIG. 3 is a map of the vector pERK-3/M/GP5.

FIG. 4 is a graph showing total gross lung score in different treatmentgroups. Treatment groups with different letters are significantlydifferent (ANOVA, p<0.05).

FIG. 5 is a graph showing interstitial pneumonia scores in differenttreatment groups. Treatment groups with different letters aresignificantly different (ANOVA, p<0.05).

FIG. 6 is a graph showing a summary of lung lymphoid hyperplasia scores.Treatment groups with different letters are significantly different(ANOVA, p<0.05).

FIG. 7 is a graph showing a summary of heart pathology scores. Treatmentgroups with different letters are significantly different (ANOVA,p<0.05).

FIG. 8 is a graph showing mean IDEXX ELISA S/P titer per group. Groupswith different letters are significantly different (ANOVA, p<0.01)within day post challenge. The bars of each group are in the orderlisted: strict negative, placebo, ARP, inacivated and MLV.

FIG. 9 is a graph showing the number of pigs out of ten per group with aFFN titer ≧4. Groups with different letters are significantly different(Chi-square, p<0.05) within day post challenge. The bars of each groupare in the order listed: strict negative, placebo, ARP, inacivated andMLV.

FIG. 10 is a graph showing geometric mean FFN titer by group. Groupswith different letters are significantly different (ANOVA, p<0.01)within day post challenge. The bars of each group are in the orderlisted: strict negative, placebo, ARP, inacivated and MLV.

FIG. 11 is a graph showing live virus titration at 7 DPC. Groups withdifferent letters are significantly different (ANOVA, p<0.01).

FIG. 12 is a graph showing the number of pigs out of ten per group PRRSVpositive serum via RT-PCR. Groups with different letters aresignificantly different (Chi-square, p<0.05) within day post challenge.The bars of each group are in the order listed: strict negative,placebo, ARP, inacivated and MLV.

FIG. 13 is a Western blot confirming recombinant HA expression. Lane 1is the ladder; lane 2 the Vero lysate (negative control); lane 3,recombinant HA (28.5 μg/ml); Lane 4: Recombinant HA (1.14 μg/ml); Lane5: Recombinant HA (0.57 μg/ml); Lane 6: Recombinant HA (0.38 μg/ml).

FIG. 14 is a graph showing a study measuring neutralizing antibodiesagainst FMDV A24 at defined days post vaccination or challenge. Thecontrol is the first bar in each grouping, Dose A is the second bar andDose B the third bar.

FIG. 15 is a graph showing serial dilution of clarified inoculum dilutedin 2% saline. Shaminoculation received an equivalent dose of 2% saline.N=20 shrimp per treatment.

FIG. 16 is a graph showing results of Example 4, Experiment 1. Shrimpwere injected with 2 ug of dsRNA construct and challenged with IMNV 48hours following administration. N=3 groups of 20 shrimp per treatment.

FIG. 17 is a graph showing results of Example 4, Experiment 2. Shrimpwere inoculated with a serial dilution of dsRNA and challenged 48 hoursfollowing administration. N=10 shrimp per treatment.

FIG. 18 is a graph showing results of Example 4, Experiment 2. Shrimpwere injected with a serial dilution of dsRNA and challenged 10 daysfollowing administration. N=10 shrimp per treatment.

FIG. 19A is a graph showing results of Example 4, Experiment 3. Shrimpwere injected with 0.02 μg of dsRNA#3, 5′ truncate of dsRNA3, or a 5′truncate of dsRNA3 and challenged 10 days following administration. N=3groups of 10 shrimp per treatment.

FIG. 19B is a graph showing results of Example 4, Experiment 3, whereshrimp were injected as in FIG. 24A, but with further truncates ofdsRNA#3.

FIG. 20 is a graph showing results of Example 4, Experiment 4 Shrimpwere inoculated with replicons or dsRNA a16nd challenged 3 daysfollowing administration. N=3 groups of 10 shrimp per treatment.

FIG. 21 is a graph showing results of Example 4, Experiment 5 Shrimpwere inoculated with replicon and challenged 3 days followingadministration. N=3 groups of 10 shrimp per treatment.

FIG. 22 is a graph showing results of Example 4, Experiment 5 Shrimpwere inoculated with replicon and challenged 10 days followingadministration. N=3 groups of 10 shrimp per treatment.

FIG. 23 is a graph showing results of Example 4, Experiment 8 WSSVsurvival following primary challenge.

FIG. 24: Survival following secondary WSSV challenge 21 days followingprimary challenge.

FIG. 25 is a graph showing survival following vaccination via injectionor reverse gavage. Animals were challenged 14 days post vaccination.

FIG. 26 is a graph showing survivorship of animals following challengethat were administered dsRNA 2 days post challenge. X-axis is days postchallenge. Y-axis is percent survival. dsRNA3 and eGFP dsRNA groups weretreated with 5 ug dsRNA and the challenge control was treated with anequivalent volume sterile water.

FIG. 27 is a graph showing percent survivorship of shrimp followingtreatment with feed containing different solutions. X-axis is days postinfection with WSSV and Y-axis is percent of animals surviving. n=30animals with 3 replicates of 10/treatment

FIG. 28 is a graph showing shrimp survival post-vaccination with dsRNAof varying lengths (day 0) and post IMNV infection (day 10). dsRNAtarget position on the IMNV genome and length are indicated in the key.

SUMMARY

Methods of producing compositions used to protect aquatic invertebretesfrom disease are disclosed. A protective molecule is produced byidentifying a target nucleic acid molecule of a microorganism causingdisease, there interference of which protects the animal from thedisease, and preparing a dsRNA molecule, an antisense RNA molecule orDNA producing same or a replicon particle comprising the same. Whenadministered to the animal, it is protected form the disease. Nucleicacid molecules of WSSV VP19 and VP28 which protect animals from thedisease are disclosed. Also disclosed are antisense and dsRNA moleculeswhich protect the animal from disease. Vaccines and compositions arealso disclosed.

DESCRIPTION

Provided here is a method of protecting aquatic invertebrates, and inparticular, shrimp, from disease. In an embodiment a delivery system isprovided of specific nucleic acid molecules that can induce a protectiveand/or immune response in the invertebrate to the pathogen.

The examples provided here show use of the invention with shrimp, and itis considered to be particularly useful in protecting shrimp (as in theclass Malacostraca which includes Decapods including Dendrobranchiatessuch as prawns and Carideans such as shrimp) from disease or disorders.However, other invertebrates and in particular aquatic invertebrates,freshwater and marine, are expected to benefit from protection fromdisease and disorder provided by the invention including, by way ofexample without limitation, crustacean (e.g. lobsters, crabs, shrimp,crayfish), mollusks (e.g., squid, clams, octopus, snails, abalone,mussels), Porifera (sponges), Cnidaria (e.g., jellyfish, sea anemones),Ctenophora, Echinodermata and aquatic worms. The invention isparticularly useful in aquatic invertebrates having commercial value,and especially useful with farmed aquatic invertebrates (as opposed tothose living in the wild at sea), as explained herein. As shown herein,it is possible to deliver the nucleic acid molecules and/or polypeptidesor fragments thereof of the invention to the digestive tract of theanimal (which can be found after administration throughout the digestivetract or a portion thereof), whether by immersion, oral delivery or thelike. This greatly aids the delivery of a vaccine to the animal, asopposed to methods such as injection, and provides a practical andeffective means of vaccinating the animals, especially with massvaccination of a multitude of animals.

The methods of the invention include means of interference withexpression of a nucleic acid molecule of the disease-causing agent ornucleic acid molecule of the disease-causing agent. When referring tointerference with expression, it is meant that expression of the nucleicacid molecule is inhibited, disrupted, or otherwise interfered with suchthat the animal is protected from the disease. In one embodiment, themethod uses an antisense RNA that is complimentary to a nucleic acidmolecule of the disease-causing agent (target nucleic acid molecule).Antisense RNA is RNA that is complementary to a target, usually amessenger RNA (mRNA) of a target nucleic acid molecule. By antisense isintended a sequence that is in inverse orientation to the 5′-to-3′normal orientation of the target nucleic acid molecule. When deliveredinto a cell, expression of the antisense RNA sequence prevents normalexpression of the protein encoded by the targeted nucleic acid molecule.When referring to RNA being a complement is meant to include that thepolynucleotide for use in antisense suppression may correspond to all orpart of the complement of the sequence encoding the target polypeptide,all or part of the complement of the 5′ and/or 3′ untranslated region ofthe target polypeptide transcript, or all or part of the complement ofboth the coding sequence and the untranslated regions of a transcriptencoding the target polypeptide. A complementary nucleic acid moleculeis that which is complementary to an mRNA transcript of all or part of atarget nucleic acid molecule. In addition, the antisense polynucleotidemay be fully complementary (i.e., 100% identical to the complement ofthe target sequence) or partially complementary (i.e., less than 100%identical to the complement of the target sequence) to the targetsequence. Antisense suppression may be used to inhibit the expression ofmultiple proteins in the same cell. Furthermore, portions of theantisense nucleotides may be used to disrupt the expression of thetarget nucleic acid molecule. Generally, antisense sequences of at least10 nucleotides, 20 nucleotides, 50 nucleotides, 100 nucleotides, 200nucleotides, 300, 500, 550, 500, 550, or greater and any amountin-between may be used. The sequence may be complementary to anysequence of the messenger RNA, that is, it may be proximal to the5′-terminus or capping site, downstream from the capping site, betweenthe capping site and the initiation codon and may cover all or only aportion of the non-coding region, may bridge the non-coding and codingregion, be complementary to all or part of the coding region,complementary to the 3′-terminus of the coding region, or complementaryto the 3′-untranslated region of the mRNA. The antisense sequence may becomplementary to a unique sequence or a repeated sequence, so as toenhance the probability of binding. Thus, the antisense sequence may beinvolved with the binding of a unique sequence, a single unit of arepetitive sequence or of a plurality of units of a repetitive sequence.Methods of preparing antisense nucleic acid molecules are known. See,e.g. Shewmaker et al, U.S. Pat. No. 5,759,829, incorporated herein byreference.

In another embodiment of the invention, RNA interference is used and ina preferred embodiment double-stranded RNA molecules (dsRNA) areemployed. In this process, in summary, RNA which is double stranded, inpart, or completely, is produced based upon the sequence of the targetnucleic acid molecule. Specifics of the means of producing the dsRNA mayvary as one skilled in the art appreciates, and include, by way ofexample without intending to be limiting, the approach of Graham et al.,U.S. Pat. No. 6,573,099 where two copies of a sequence corresponding toa target sequence is used, or that of Fire et al., U.S. Pat. No.6,326,193, where the first strand is an RNA sequence corresponding tothe target nucleic acid, and the second is one which is complementary tothe target sequence, each of which are incorporated herein by referencein their entirety. These strands hybridize with each other to form theinhibiting dsRNA. The strand which corresponds to the target nucleicacid molecule can correspond to all or a portion thereof, so long as adsRNA is formed. Where a strand is used which is the complement(antisense) of the target nucleic acid is used, it can be complementaryto all or a portion of the target nucleic acid molecule, so long as thedsRNA formed interferes with the target nucleic acid molecule. The dsRNAtriggers a response in which the RNAse III Dicer enzyme process dsRNAinto small interfering RNAs (siRNA) of approximately 21-23 nucleotides,which are formed into a RNA-induced silencing complex RISC whichdestroys homologous mRNAs. (See, Hammond, S. M., et al., Nature (2000)404:293-296). When referring to a target nucleic acid molecule it ismeant a nucleic acid molecule or fragment thereof of the disease agent,the expression of which is interfered with. Generally, sequences of atleast 10 nucleotides 20 nucleotides, 30 nucleotides, 40 nucleotides, 50nucleotides, 100 nucleotides, 200 nucleotides, 300, 500, 550, 500, 550,or greater and any amount in-between may be used.

The inventors have shown examples of dsRNA sequences which can be usedin the invention, and have discovered that fragments of such dsRNA canbe used to provide a protective response. For example, dsRNA#3 thatinterferes with IMNV and provides a protective response is a 380 basepair sequence. However, fragments of the dsRNA provide a protectiveresponse. Thus when referring to dsRNA of the invention, fragments ofthe dsRNA that provide such a protective response are included.

As discussed below, the inventors have also demonstrated that a nucleicacid molecule encoding a polypeptide or fragment thereof of thedisease-causing agent may be administered to the aquatic invertebrateanimal and a protective response observed.

In an embodiment, Replicon Particle technology is used to deliver theprotective molecule to the animal. The nucleic acid molecule,polypeptide encoded, or fragments of the nucleic acid molecule orpolypeptide, or the antisense or dsRNA are those which produce aprotective response when administered to the animal and are referred toherein at times as the protective molecule. The protective molecule isintroduced into a cell by any of the various means available to oneskilled in the art, whether by uptake, absorption, through cellulaseprocesses, or auxiliary agents or devices, injection or the like,examples of which are described below.

In one embodiment the vaccine of the invention comprises the protectivemolecule. In another embodiment the vaccine is made by producing thedsRNA which can then be introduced directly into the aquaticinvertebrate animal cell, or placed in a vector or expression cassetteand introduced into the cell. The inventors have discovered that thedsRNA can be introduced directly into the cell and a protective responseis produced. The dsRNA could be delivered by a DNA vector that thenproduces the dsRNA from a promoter that is recognized by some cellularDNA-dependant RNA polymerase. In another embodiment, Replicon Particletechnology may be employed in producing the vaccine. Where the antisenseRNA is used as a vaccine, without wishing to be bound by any theory, itis believed it then forms a dsRNA in the cell into which it isintroduced.

Prior to introducing the protective molecule, one identifies a nucleicacid sequence in the disease-causing agent which is to be expressed orinhibited (target nucleic acid molecule or target gene). The protectivemolecules may either express, inhibit, or compete for binding sites withany such target nucleic acid molecule which, when administered, resultsin protection to the animal from the disease causing agent. Any suchprotective molecule may be employed in the invention. Examples, withoutintending to be limiting of such protective molecules are those encodingor inhibiting White Spot Syndrome Virus (WSSV) or fragments thereof, andin a preferred embodiment, encoding or inhibiting VP28, and VP19polypeptides of WSSV or fragments thereof, or Infectious MyonecrosisVirus (IMNV) or fragments thereof and which stimulate a protectiveresponse. The inventors have also shown in one embodiment that aReplicon Particle expressing VP19 antisense and VP19 protein providesprotection. Further, fragments of dsRNAs of target molecules are shownhere to also provide protection.

Once that genetic information is obtained, a nucleic acid molecule or anantisense or dsRNA of such target nucleic acid molecule is provided as avaccine.

In one embodiment, the “naked” nucleic acid molecules or naked dsRNA orantisense molecule may be administered to the aquatic animal, that isthe dsRNA or antisense need not be provided in a conventional expressioncassette or vector. Such a molecule may be produced by any convenientmethod, such as primer amplification and reverse transcription such asis described below.

In another embodiment, the protective molecule may be delivered by anexpression cassette or vector which may optionally include othercomponents. In a further embodiment, the protective molecule may bedelivered by Replicon Particle. In yet another embodiment, delivery ofthe vaccine to the digestive tract of the animal provides protection.

A “vector” is any means for the transfer of a nucleic acid into a hostcell. A vector may be a replicon to which a DNA segment may be attachedso as to bring about the replication of the attached segment. A“replicon” is any genetic element (e.g., plasmid, phage, cosmid,chromosome, virus) that functions as an autonomous unit of DNA or RNAreplication in vivo, i.e., capable of replication under its own control.The term “vector” includes both viral and nonviral means for introducingthe nucleic acid into a cell in vitro, ex vivo or in vivo. Viral vectorsinclude alphavirus, retrovirus, adeno-associated virus, pox,baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirusvectors. Non-viral vectors include, but are not limited to plasmids,liposomes, electrically charged lipids (cytofectins), DNA- or RNAprotein complexes, and biopolymers. In addition to a nucleic acid, avector may also contain one or more regulatory regions, and/orselectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (transfer to which tissues, duration ofexpression, etc.).

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest or produces RNA, and the cassette andrestriction sites are designed to ensure insertion of the cassette inthe proper reading frame for transcription and translation.

A nucleic acid molecule is introduced into a cell when it is inserted inthe cell. A cell has been “transfected” by exogenous or heterologous DNAor RNA when such DNA or RNA has been introduced inside the cell.

A cell has been “transformed” by exogenous or heterologous DNA when thetransfected DNA effects a phenotypic change. The transforming DNA can beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given polypeptide. For instance, the codons CGU, CGC,CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, atevery position where an arginine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentsubstitutions” or “silent variations,” which are one species of“conservatively modified variations.” Every polynucleotide sequencedescribed herein which encodes a polypeptide also describes everypossible silent variation, except where otherwise noted. Thus, silentsubstitutions are an implied feature of every nucleic acid sequencewhich encodes an amino acid. One of skill will recognize that each codonin a nucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified to yield a functionally identical moleculeby standard techniques. In some embodiments, the nucleotide sequencesthat encode a protective polypeptide are preferably optimized forexpression in a particular host cell (e.g., yeast, mammalian, plant,fungal, and the like) used to produce the polypeptide or RNA.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” referred to herein as a “variant”where the alteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.See, for example, Davis et al., “Basic Methods in Molecular Biology”Appleton & Lange, Norwalk, Conn. (1994). Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, 1984, Proteins).

The isolated variant proteins can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Forexample, a nucleic acid molecule encoding the variant polypeptide iscloned into an expression vector, the expression vector introduced intoa host cell and the variant protein expressed in the host cell. Thevariant protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.

A protein is comprised of an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein may be a the originalpolypeptide, a variant polypeptide and/or have additional amino acidmolecules, such as amino acid residues (contiguous encoded sequence)that are naturally associated with it or heterologous amino acidresidues/peptide sequences. Such a protein can have a few additionalamino acid residues or can comprise several hundred or more additionalamino acids.

The variant proteins used in the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a variant protein fused in-frameto a heterologous protein having an amino acid sequence notsubstantially homologous to the variant protein. The heterologousprotein can be fused to the N-terminus or C-terminus of the variantprotein.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A variant protein-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the variant protein.

Polypeptides sometimes contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in polypeptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art.Accordingly, the variant peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence for purification of themature polypeptide or a pro-protein sequence.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cros slinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

The present invention further provides fragments of the variant proteinsof the present invention, in addition to proteins and peptides thatcomprise and consist of such fragments, provided that such fragments actas an antigen and/or provide treatment for and/or protection againstinfections as provided by the present invention.

The phrase “biological sample” refers to a fluid or tissue of an animal.Such components are known in the art and include, without limitation,blood, plasma, serum, and secretions of the intestinal or genitourinarytracts.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced byan organism in response to an antigen challenge. The antibodies usedwith the present invention include monoclonal antibodies and polyclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)hd 2, and Fv fragments.

As used herein, the term “subunit” refers to a portion of themicroorganism which may itself antigenic, i.e., capable of inducing animmune response in an animal or protective. The term should be construedto include subunits which are obtained by both recombinant andbiochemical methods.

As used herein, the term “isolate” refers to a virus obtained from aspecific source. Isolate is used interchangeably with the term “strain”.

As used herein, the term “vaccine” as used herein refers to apharmaceutical composition comprising at least one protective molecule,nucleic acid or polypeptide or fragment thereof that induces animmunological and/or protective response in an animal and possibly, butnot necessarily, one or more additional components that enhance theactivity of said active component. A vaccine may additionally comprisefurther components typical to pharmaceutical compositions. In anotherform, the immunologically active component of a vaccine may compriseappropriate elements of said organisms (subunit vaccines) whereby theseelements are generated either by destroying the whole organism or thegrowth cultures of such microorganisms and subsequent purification stepsyielding in the desired structure(s), or by synthetic processes inducedby an appropriate manipulation of a suitable system such as, but notrestricted to, bacteria, insects, mammalian, or other species, plussubsequent isolation and purification procedures or by induction of saidsynthetic processes in the animal needing a vaccine by directincorporation of genetic material using suitable pharmaceuticalcompositions (polynucleotide vaccination). A vaccine may comprise one orsimultaneously more than one of the elements described above.

The terms “protecting”, “protection”, “protective immunity” or“protective immune response,” as used herein, are intended to mean thatthe host animal mounts an active immune response to the vaccine orpolypeptides of the present invention, such that upon exposure to thedisease challenge, the animal is able to combat the infection. Thus, aprotective immune response will decrease the incidence of morbidity andmortality from exposure to the microorganism among a host animal. Theanimal will be protected from subsequent exposure to the disease-causingagent. In an embodiment, the animal may be protected by treating theanimal which has already been exposed to the disease-causing agent byadministration of the vaccine or polypeptide after such exposure. Insuch an instance there is also shown to be a lessening of morbidity andmortality. Those skilled in the art will understand that in a commercialanimal setting, the production of a protective immune response may beassessed by evaluating the effects of vaccination on a pond, group,flock or herd as a whole, e.g., there may still be morbidity andmortality in a minority of vaccinated animals. Furthermore, protectionalso includes a lessening in severity of any gross or histopathologicalchanges and/or of symptoms of the disease, as compared to those changesor symptoms typically caused by the isolate in similar animals which areunprotected (i.e., relative to an appropriate control). Thus, aprotective immune response will decrease the symptoms of the disease,which will vary according to the disease. Those skilled in the art willalso understand that in the case of an arthropod host, protectiveimmunity does not necessarily equate to the traditional memory responsecharacteristic of adaptive immunity in vertebrate animals. Diseasemorbidity and/or mortality is reduced and where there also may be areduced titer of infection upon exposure to the microorganism.

As used herein, “immunogenically effective amount” refers to an amount,which is effective in reducing, eliminating, treating, preventing orcontrolling the symptoms of the infections, diseases, disorders, orcondition.

In one embodiment, the present invention relates to a polypeptidecomprising a polypeptide or fragment thereof of microorganism. Thepresent inventors contemplate that the polypeptide may be a homologue, aderivative, or a variant of the polypeptide, or an immunologicallyactive or a functional fragment thereof. The polypeptide may beisolated, synthesized, or recombinantly expressed using thepolypeptide-encoding nucleic acids described herein.

The present invention also provides isolated and/or recombinant nucleicacids that encode a polypeptide or RNA of the invention. In addition, itshould be understood based on the general state of the art that otherequivalent sequences to the nucleotide or amino acid sequences of thepolypeptides are covered by the present invention. For example, somedeletions, insertions and substitutions in the amino acid sequenceisolated from the microorganism or expressed by a nucleic acid sequenceisolated from the microorganism are covered by the present invention,unless such mutation abolishes the ability of the polypeptide to inducethe generation of protective response.

Nucleic acids of the invention include those that encode an entirepolypeptide or produce an RNA sequence as well as those that encode asubsequence of the polypeptide or RNA or produce a fragment of a dsRNA.For example, the invention includes nucleic acids that encode apolypeptide or RNA which is not full-length but nonetheless hasprotective activity against infection. The invention includes not onlynucleic acids that include the nucleotide sequences as set forth herein,but also nucleic acids that are substantially identical to, correspondto, or substantially complementary to, the exemplified embodiments. Forexample, the invention includes nucleic acids that include a nucleotidesequence that is at least about 70% identical to one that is set forthherein, more preferably at least 75%, still more preferably at least80%, more preferably at least 85%, 86%, 87%, 88%, 89% still morepreferably at least 90%, 91%, 92%, 93%, 94%, and even more preferably atleast about 95%, 96%, 97%, 98%, 99%, 100% identical (or any percentagein between) to an exemplified nucleotide sequence. The nucleotidesequence may be modified as described previously, so long anypolypeptide encoded or RNA or dsRNA produced is capable of inducing thegeneration of a protective response.

The inventors have shown that dsRNA sequences produced can includetruncated fragments. Such fragments can be 9 or more base pairs, can be10 base pairs, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 72, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 base pairsor more or any number in-between, as long as a protective response isseen when administered to an animal. According to an embodiment of theinvention, without wishing to be bound by any theory, at least ninebases are used to provide for sequence specificity such thatinterference with the target molecule occurs. A well-described mechanismof action for RNA interference (RNAi) is described for cellularmicroRNAs (miRNA) that is related to the action noted for the dsRNAsdescribed above. The 5′-most seven to 8 nucleotides of a miRNA(sometimes referred to as the seed sequence) are involved inWatson-Crick base pairing with nucleotides in the 3′ untranslated regionof the target mRNA. (Lewis, B. P., C. B. Burge, and D. P. Bartel. 2005.Conserved seed pairing, often flanked by adenosines, indicates thatthousands of human genes are miRNA targets Cell 120:15-20.) RNA-inducedsilencing complex (RISC) cleaves target mRNA where base pairing isperfect, and where imperfect, the target mRNA is translationallyinactive, and protein expression is impacted without degrading mRNA.(Bartel, D. P. 2004. MicroRNAs: genomics, biogenesis, mechanism, andfunction. Cell. 116:281-97.) It is likely that the same RISC-basedactivity is the mechanism by which the dsRNA described above areproviding protection to shrimp. In a preferred embodiment, at least 20bases are used, and in another preferred embodiment at least 30 basesare used, which further aids in transport into the cells. In anotherfurther preferred embodiment, higher efficacy is achieved with a dsRNAthat is at least 50 bp or more in length.

To test such fragments, a process employment AEM (antiviral effectormolecule) development is one of many available methods. According tosuccesses in AEM development for IMNV, this process generally proceedsas follows: a long target region for AEM development is determined,protection from disease measured according to survival post challengewith the established disease challenge model, shorter length dsRNAs willbe assessed in standard disease bioassays (described by way of examplein Examples 1, 2 and 3 below) by designing dsRNAs to progressivelyshorter target regions within the proven, longer length AEM.

For each target of interest, in an example, a set of PCR primers with 5′T7 promoter sequence is designed to produce ˜400 bp portions of theamplicon sequence AS. Amplicon sequence that is encompassed by the PCRprimers are filtered by screening against the genome and transcriptomesequences to predict and minimize potential off-target effects. PCRproducts are generated from whole body cDNA derived from pooled larvaland adult RNA. From this product, dsRNAs are produced using the AmbionMega script in vitro transcription kit and yields 50-100 μg of highquality dsRNA per reaction. Typically, dsRNA yields of 50-100 ug areachieved from a single in vitro transcription reaction. It should benoted that the entire process of generating dsRNA production, fromgenerating gene specific primers with 5′ T7 promoter sequence, toproduct is usually 3 days (including primer synthesis and O/N shipping).Heterologous dsRNA to eGFP is used to control for the physiologic impactof triggering a dsRNA response. This process is repeated withsuccessively truncated regions of proven targets. Gene suppression ismeasured by any available method, including quantitative RT-PCR orRT-PCR, nucleic acid hybridization or Northern blotting of whole body orspecific tissues RNA extracts, using primer sets that extend beyond orare completely removed from the region encompassed by the dsRNAgenerating primer set.

The nucleic acids that encode a polypeptide or produce an RNA providinga protective response can be obtained using methods that are known tothose of skill in the art. Suitable nucleic acids (e.g., cDNA, genomic,or subsequences) can be cloned, or amplified by in vitro methods such asthe polymerase chain reaction (PCR) using suitable primers, the ligasechain reaction (LCR), the transcription-based amplification system(TAS), the self-sustained sequence replication system (SSR). A widevariety of cloning and in vitro amplification methodologies arewell-known to persons of skill Examples of these techniques andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology 152 Academic Press, Inc., San Diego,Calif. (Berger); Sambrook et al. (2001) Molecular Cloning—A LaboratoryManual (Third ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor Press, NY, (Sambrook et al.); Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1994 Supplement) (Ausubel); Cashion et al., U.S. Pat. No. 5,017,478;and Carr, European Patent No. 0,246,864. Examples of techniquessufficient to direct persons of skill through in vitro amplificationmethods are found in Berger, Sambrook, and Ausubel, as well as Mullis etal., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methodsand Applications (Innis et al., eds) Academic Press Inc. San Diego,Calif. (1990) (Innis); Amheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3: 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35: 1826; Landegrenet al., (1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4: 560; and Barringer et al. (1990)Gene 89: 117. Improved methods of cloning in vitro amplified nucleicacids are described in Wallace et al., U.S. Pat. No. 5,426,039. Nucleicacids that encode the polypeptide or RNA of the invention, orsubsequences of these nucleic acids, can be prepared by any suitablemethod as described above, including, for example, cloning andrestriction of appropriate sequences.

“Codon optimization” can be used to optimize sequences for expression inan animal and is defined as modifying a nucleic acid sequence forenhanced expression in the cells of the aquatic animal of interest, e.g.shrimp, by replacing at least one, more than one, or a significantnumber, of codons of the native sequence with codons that are morefrequently or most frequently used in the genes of that invertebrate.Various species exhibit particular bias for certain codons of aparticular amino acid.

In one aspect, the present invention relates to polynucleotidescomprising nucleic acid fragments of codon-optimized coding regionswhich encode polypeptides or produce RNA, or fragments, variants, orderivatives thereof, with the codon usage adapted for optimizedexpression in the cells of a given aquatic animal. These are prepared byincorporating codons preferred for use in the genes of the animal ofinterest into the DNA sequence. Also provided are constructs, vectors,and host cells comprising nucleic acid fragments of codon-optimizedcoding regions, and fragments, variants, or derivatives thereof, andvarious methods of using the polynucleotide expression constructs,vectors, host cells to treat or prevent disease in an animal.

A nucleic acid encoding a polypeptide or producing RNA may then beintroduced into either a prokaryotic or eukaryotic host cell through theuse of a vector, plasmid or construct and the like to produce thepolypeptide. A typical expression cassette contains a promoter operablylinked to a nucleic acid that encodes the product of interest. Theexpression cassettes are typically included on expression vectors thatare introduced into suitable host cells, including for example,bacterial, insect, fungal, plant or animal cells. Either constitutive orregulated promoters can be used in the present invention. Promoterssuitable for use in eukaryotic host cells are well known to those ofskill in the art. The expression vectors of the invention can betransferred into the chosen host cell by methods known to those ofordinary skill in the art including, for example, calcium phosphatetransfection, DEAE-dextran mediated transfection, transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. (See Molecule Cloning: A Laboratory Manual, 2d Ed. Vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)).Transformed cells can be selected, for example, by resistance toantibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

In an example, the protective molecule may be expressed by a recombinantvector, viral vector, or virus. In another aspect, the recombinantvector, viral vector, or microorganism expressing the protectivemolecule may itself serve as a vaccine component acting as a as anprotective agent or an adjuvant and eliciting or enhancing theprotective response. By way of example, suitable recombinant virusvectors include but are not limited to live adenovirus, poxvirus,baculovirus, pseudorabies virus (PRV), Venezuelan equine encephalitis(VEE) vectors such as strains V3526 or TC-83, and viral repliconparticles (VRPs) derived from VEE, equine arteritis virus (EAV), ortransmissible gastroenteritis virus (TGE). The techniques employed toinsert such a sequence into the viral vector and make ether alterationsin the viral DNA, e.g., to insert linker sequences and the like, areknown to one of skill in the art. (See, e.g., Molecular Cloning. ALaboratory Manual, supra.)

The nucleic acid molecule may be operably linked to a suitable promoterat the 5′ end of the cDNA encoding a polypeptide or producing RNA and atermination signal and poly(A) signal at the 3′ end of the cDNA. As usedherein, the term “operably linked” means that the nucleic acid moleculecontaining an expression control sequence, e.g., transcription promoterand termination sequences, are situated in a vector or cell such thatexpression of the polypeptide or RNA produced by the nucleic acidmolecule is regulated by the expression control sequence. Methods forcloning and operably linking such sequences are well known in the art.Examples of promoters suitable for expressing the antigen include butare not limited to are the cytomegalovirus immediate-early (CMV)promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR)promoter, the simian virus 40 (SV40) immediate-early promoter, andinducible promoters such as the metallothionein promoter. Other examplesof promoters include, T7 phage promoter, T3 phage promoter,beta-galactosidase promoter, and the Sp6 phage promoter. An example of aDNA having a termination and poly(A) signal is the SV40 late poly(A)region. Another example of a viral expression system suitable forproducing the antigen is the Sindbis Expression system available fromInvitrogen. The use of these commercially available expression vectorsand systems are well known in the art.

The vaccine of the present invention may also contain multiple copies ofone protective molecule or a combination of protective molecules.

In another embodiment, replicon particle (RP) vaccines are prepared. TheRP vector has numerous advantages for vaccine development includingaccurate production of native proteins, tropism for lymphoid cells, lackof viral replication and transmission, induction of mucosal and systemicimmunity, sequential immunization potential, and lack of preexistingimmunity to VEE in animals although they clearly can respond to thevirus immunologically. (Dickerman R W, Baker G J, Ordonez J V, Cherer WF (1973), Venezuelan Equine Encephalomyelitis Viremia and AntibodyResponses of Pigs and Cattle, American Journal of Veterinary Research34: 357-361.)

The replication strategy of VEE is similar to that of otheralphaviruses. (Strauss J, Strauss E (1994), The alphaviruses: geneexpression, replication, and evolution, Microbiol Rev 58: 491-562.) Frompositive-sense genomic RNA, four non-structural proteins (nsP1-nsP4) aretranslated and function to replicate a full-length negative-sense RNA.The negative-sense RNA serves as a template for replication ofadditional genomic RNA, and for synthesis of a subgenomic messenger RNA(26S mRNA), produced in 10-fold molar excess compared to genomic RNA,which directs the synthesis of the VEE structural proteins. Thestructural proteins are translated initially as a polyprotein that isco-translationally and post-translationally cleaved to release thecapsid (C) protein and the two mature envelope glycoproteins (E1 andE2). Since VEE is a positive-sense RNA virus, full-length cDNA clones ofVEE can be used to generate RNA transcripts that, when introduced intosusceptible cells, will initiate a complete virus replication cycle andgenerate infectious virus. (Davis N L, Willis L V, Smith J F, Johnston RE (1989), In vitro synthesis of infectious Venezuelan equineencephalitis virus RNA from a cDNA clone: analysis of a viable deletionmutant, Virology 171: 189-204.)

Using site-directed mutagenesis of the DNA plasmid, VEE viruses can begenerated containing mutations in the envelope glycoproteins that resultin attenuated phenotypes. When inoculated into animals, such attenuatedvariants of VEE do not cause illness or significant viremia but are ableto induce protective immunity against subsequent virulent VEE challengein mice, horses and primates. (Davis N, Powell N, Greenwald G, Willis L,Johnson B, Smith J, Johnston R (1991), Attenuating mutations in the E2glycoprotein gene of Venezuelan equine encephalitis virus: constructionof single and multiple mutants in a full-length cDNA clone, Virology183: 20-31; Grieder F, Davis N, Aronson J, Charles P, Sellon D, SuzukiK, Johnston R (1995), Specific restrictions in the progression ofVenezuelan equine encephalitis virus-induced disease resulting fromsingle amino acid changes in the glycoproteins, Virology 206: 994-1006.)

Similarly, foreign genes can be inserted in place of the VEE structuralprotein gene region in the cDNA plasmid, and an RNA transcript from sucha plasmid, when introduced into cells, will replicate and express theheterologous genes. This self-amplifying replicon RNA will direct thesynthesis of large amounts of the foreign gene product within the cell,typically reaching levels of 15-20% of total cell protein. (Pushko P,Parker M, Ludwig G V, Davis N, Johnston R E, Smith J F (1997),Replicon-Helper Systems from Attenuated Venezuelan Equine EncephalitisVirus: Expression of Heterologous Genes in Vitro and Immunizationagainst Heterologous Pathogens in Vivo, Journal of Virology 239:389-401.)

Because the replicon RNA does not contain the structural genes for VEE,it is a single-cycle, propagation-defective RNA and replicates onlywithin the cell into which it is introduced. The replicon RNA can bepackaged into RP by supplying the structural protein genes of VEE intrans (FIGS. 1A and 1B). Replicon RNA is packaged into RP when cells areco-transfected with replicon RNA and two separate helper RNAs, whichtogether encode the full complement of VEE structural proteins. Pushko,supra. Importantly, only the replicon RNA is packaged into VRP, as thehelper RNAs lack the packaging sequence required for encapsidation.Thus, the RP are propagation-defective, in that they can infect targetcells in culture or in vivo, can express the foreign gene to highlevels, but they lack critical portions of the VEE genome (i.e., the VEEstructural protein genes) necessary to produce virus particles whichcould spread to other cells. The “split helper” system greatly reducesthe chance of an intact genome being regenerated by RNA-RNArecombination and the possibility of functional recombination withhelper RNAs was further reduced by removal of the 26S promoter fromhelper RNAs altogether (Kamrud et al 2010 Development andCharacterization of Promoterless Helper RNAs for Production ofAlphavirus Replicon Particles. Journal of General Virology. 91:pp.1723-1727.). As an independent and additional layer of safety;attenuating mutations have been incorporated in the glycoprotein helper(Pushko et al 1997 Journal of Virology 239:389-401). FIG. 6 shows theVEE replicon particle vaccine and packaging system process. Expressionof the nucleic acid molecule of interest can be varied up or down byintroducing spacer elements upstream of the IRES/NOI cassette butdownstrem of the 26S promoter. (Kamrud et al. 2007, “Alphavirus RepliconApproach to Promoterless Analysis of IRES Elements”, Virology 360(2),pp. 376-387) Also, where the gene of interest produces a potentiallytoxic protein, introducing a phosphoramidite morpholino oligomers at thesame time the replicon and helper RNAs are electroporated into cellsshuts down expression. The PMO blocks translation of the gene ofinterest during packaging of RP.

The methods and variations of same used to produce such replicons areknown to one skilled in the art. Illustrative methodology can be foundat U.S. Pat. No. 6,156,558, incorporated herein by reference in itsentirety, and also at U.S. Pat. Nos. 6,521,235; 6,531,135; and U.S. Pat.Nos. 7,442,381; 6,541,010; 7,045,335; and 5,792,462 all of which areincorporated herein by reference in their entirety.

Alphavirus vectors and alphavirus replicon particles are used inembodiments of the invention. The term “alphavirus” has its conventionalmeaning in the art, and includes the various species of alphaviruseswhich are members of the Togaviridae family. This includes alphavirusessuch as Eastern Equine Encephalitis virus (EEE), Venezuelan EquineEncephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus,Western Equine Encephalitis virus (WEE), Sindbis virus, South AfricanArbovirus No. 86, Semliki Forest virus, Middelburg virus, Chikungunyavirus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getahvirus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Auravirus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands Jvirus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The viralgenome is a single-stranded, messenger-sense RNA, modified at the 5′-endwith a methylated cap, and at the 3′-end with a variable-length poly (A)tract. Structural subunits containing a single viral protein, C,associate with the RNA genome in an icosahedral nucleocapsid. In thevirion, the capsid is surrounded by a lipid envelope covered with aregular array of transmembranal protein spikes, each of which consistsof a heterodimeric complex of two glycoproteins, E1 and E2. See Pedersenet al., J. Virol. 14:40 (1974). The Sindbis and Semliki Forest virusesare considered the prototypical alphaviruses, and have been studiedextensively. See Schlesinger The Togaviridae and Flaviviridae, PlenumPublishing Corp., New York (1986). The VEE virus has also been studied.See U.S. Pat. No. 5,185,440 to Davis et al.

As the above patents illustrate, preparation of replicon subunits byusing alphavirus replicon vectors to obtain polypeptides and usingalphavirus replicon particles to produce protective molecules areprocesses known to one skilled in the art. There are many modificationto the process available, and any process using a replicon subunit orreplicon particle methodology can be used with the invention. In acertain embodiment an alphavirus replicon RNA vector that expresses thegene of interest in a host cell and the expressed product is harvested.In another embodiment a replicon RNA comprising the gene of interest isintroduced into a cell along with two helper RNAs coding for thealphavirus capsid protein and for the glycoproteins. The replicon RNA ispackaged into a Replicon Particle. This Replicon Particle can be theprotective molecule.

Thus the system in one embodiment provides for infectious, defective,alphavirus particles, wherein each particle comprises an alphavirusreplicon RNA, and wherein the replicon RNA comprises an alphaviruspackaging signal, one or more heterologous RNA sequence(s), and asequence encoding at least one alphavirus structural protein, andwherein the replicon RNA furthermore lacks a sequence encoding at leastone alphavirus structural protein; wherein the population contains nodetectable replication-competent alphavirus particles as determined bypassage on permissive cells in culture. For example, in U.S. Pat. No.6,531,135, incorporated herein by reference in its entirety is shown inan embodiment an RP system which uses a helper cell for expressing aninfectious, replication defective, alphavirus particle in analphavirus-permissive cell. The helper cell includes (a) a first helperRNA encoding (i) at least one alphavirus structural protein, and (ii)not encoding at least one alphavirus structural protein; and (b) asecond helper RNA separate from the first helper RNA, the second helperRNA (i) not encoding at least one alphavirus structural protein encodedby the first helper RNA, and (ii) encoding at least one alphavirusstructural protein not encoded by the first helper RNA, such that all ofthe alphavirus structural proteins assemble together into alphavirusparticles in the cell. Preferably, the alphavirus packaging segment isdeleted from at least the first helper RNA.

There are many variations that are available to one skilled in the artwhen preparing such replicons. For example, in another embodimentdescribed in the patent, the helper cell also includes a replicon RNA,which encodes the alphavirus packaging segment and an insertedheterologous RNA. In the embodiment wherein the helper cell alsoincludes a replicon RNA, the alphavirus packaging segment may be, andpreferably is, deleted from both the first helper RNA and the secondhelper RNA. For example, in the embodiment wherein the helper cellincludes a replicon RNA encoding the alphavirus packaging segment and aninserted heterologous RNA, the first helper RNA includes the alphavirusE1 glycoprotein and the alphavirus E2 glycoprotein, and the secondhelper RNA includes the alphavirus capsid protein. The replicon RNA,first helper RNA, and second helper RNA in an embodiment are all onseparate molecules and are cotransfected into the host cell.

In an alternative embodiment, the helper cell includes a replicon RNAencoding the alphavirus packaging segment, an inserted heterologous RNA,and the alphavirus capsid protein encoded by the second helper RNA, andthe first helper RNA includes the alphavirus E1 glycoprotein and thealphavirus E2 glycoprotein. Thus, the replicon RNA and the first helperRNA are on separate molecules, and the replicon RNA and the secondhelper RNA are on a single molecule. The heterologous RNA comprises aforeign RNA.

The RNA encoding the structural proteins, i.e., the first helper RNA andthe second helper RNA, may advantageously include one or moreattenuating mutations. In an embodiment, at least one of the firsthelper RNA and the second helper RNA includes at least one attenuatingmutation. The attenuating mutations provide the advantage that in theevent of RNA recombination within the cell, the coming together of thestructural and non-structural genes will produce a virus of decreasedvirulence.

As another aspect a method of making infectious, replication defective,alphavirus particles is provided. The method includes transfecting ahelper cell as given above with a replication defective replicon RNA,producing the alphavirus particles in the transfected cell, and thencollecting the alphavirus particles from the cell. The replicon RNAencodes the alphavirus packaging segment and a heterologous RNA. Thetransfected cell further includes the first helper RNA and second helperRNA as described above.

As another aspect, a set of RNAs is provided for expressing aninfectious, replication defective alphavirus. The set of RNAs comprises,in combination, (a) a replicon RNA encoding a promoter sequence, aninserted heterologous RNA, wherein RNA encoding at least one structuralprotein of the alphavirus is deleted from the replicon RNA so that thereplicon RNA is replication defective, and (b) a first helper RNAseparate from the replicon RNA, wherein the first helper RNA encodes intrans, the structural protein which is deleted from the replicon RNA andwhich may or may not include a promoter sequence. In this embodiment, itis preferred that an RNA segment encoding at least one of the structuralproteins is located on an RNA other than the first helper RNA. Thus, forexample, the set of RNAs may include a replicon RNA including RNA whichencodes the alphavirus packaging sequence, the inserted heterologousRNA, and the alphavirus capsid protein, but both the alphavirus E1glycoprotein and alphavirus E2 glycoprotein are deleted therefrom; and afirst helper RNA includes RNA encoding both the alphavirus E1glycoprotein and the alphavirus E2 glycoprotein.

In another embodiment, the set of RNAs also includes a second helper RNAseparate from the replicon RNA and the first helper RNA. In thisembodiment, the second helper RNA encodes, in trans, at least onestructural protein, which is different from the structural proteinencoded by the replicon RNA and by the first helper RNA. Thus, forexample, the set of RNAs may include a replicon RNA including RNA whichencodes the alphavirus packaging sequence, and the inserted heterologousRNA; a first helper RNA including RNA which may encode a promotersequence and an RNA encoding both the alphavirus E1 glycoprotein and thealphavirus E2 glycoprotein; and a second helper RNA including RNA whichencodes the alphavirus capsid protein, with the replicon RNA, the firsthelper RNA, and the second helper RNA being in trans from each other, onseparate molecules.

As another aspect, is provided a pharmaceutical formulation comprisinginfectious alphavirus particles as described above, in an effectiveimmunogenic amount in a pharmaceutically acceptable carrier. See, forexample, the '135 patent at column 2, line 10-column 11 line 52 whichincludes examples 1-5.

The phrases “structural protein” or “alphavirus structural protein” asused herein refer to the encoded proteins which are required forproduction of particles that contain the replicon RNA, and include thecapsid protein, E1 glycoprotein, and E2 glycoprotein. As describedhereinabove, the structural proteins of the alphavirus are distributedamong one or more helper RNAs (i.e., a first helper RNA and a secondhelper RNA). In addition, one or more structural proteins may be locatedon the same RNA molecule as the replicon RNA, provided that at least onestructural protein is deleted from the replicon RNA such that thereplicon and resulting alphavirus particle are replication defective. Asused herein, the terms “deleted” or “deletion” mean either totaldeletion of the specified segment or the deletion of a sufficientportion of the specified segment to render the segment inoperative ornonfunctional, in accordance with standard usage. See, e.g., U.S. Pat.No. 4,650,764 to Temin et al. The term “replication defective” as usedherein, means that the replicon RNA cannot produce particles in the hostcell in the absence of the helper RNA. That is, no additional particlescan be produced in the host cell. The replicon RNA is replicationdefective inasmuch as the replicon RNA does not include all of thealphavirus structural proteins required for production of particlesbecause at least one of the required structural proteins has beendeleted therefrom.

The helper cell for production of the infectious, replication defective,alphavirus particle comprises a set of RNAs, as described above. The setof RNAs principally include a first helper RNA and a second helper RNA.The first helper RNA includes RNA encoding at least one alphavirusstructural protein but does not encode all alphavirus structuralproteins. In other words, the first helper RNA does not encode at leastone alphavirus structural protein; that is, at least one alphavirusstructural protein gene has been deleted from the first helper RNA. Inone embodiment, the first helper RNA includes RNA encoding thealphavirus E1 glycoprotein, with the alphavirus capsid protein and thealphavirus E2 glycoprotein being deleted from the first helper RNA. Inanother embodiment, the first helper RNA includes RNA encoding thealphavirus E2 glycoprotein, with the alphavirus capsid protein and thealphavirus E1 glycoprotein being deleted from the first helper RNA. In athird, preferred embodiment, the first helper RNA includes RNA encodingthe alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, withthe alphavirus capsid protein being deleted from the first helper RNA.The second helper RNA includes RNA encoding the capsid protein which isdifferent from the structural proteins encoded by the first helper RNA.In the embodiment wherein the first helper RNA includes RNA encodingonly the alphavirus E1 glycoprotein, the second helper RNA may includeRNA encoding one or both of the alphavirus capsid protein and thealphavirus E2 glycoprotein which are deleted from the first helper RNA.In the embodiment wherein, the first helper RNA includes RNA encodingonly the alphavirus E2 glycoprotein, the second helper RNA may includeRNA encoding one or both of the alphavirus capsid protein and thealphavirus E1 glycoprotein which are deleted from the first helper RNA.In the embodiment wherein the first helper RNA includes RNA encodingboth the alphavirus E1 glycoprotein and the alphavirus E2 glycoprotein,the second helper RNA may include RNA encoding the alphavirus capsidprotein which is deleted from the first helper RNA.

In one embodiment, the packaging segment or “encapsidation sequence” isdeleted from at least the first helper RNA. In a preferred embodiment,the packaging segment is deleted from both the first helper RNA and thesecond helper RNA.

In an embodiment wherein the packaging segment is deleted from both thefirst helper RNA and the second helper RNA, preferably the helper cellcontains a replicon RNA in addition to the first helper RNA and thesecond helper RNA. The replicon RNA encodes the packaging segment and aninserted heterologous RNA. The inserted heterologous RNA may be RNAencoding a protein or a peptide. The inserted heterologous RNA mayencode a protein or a peptide which is desirously expressed by the host,alphavirus-permissive cell, and includes the promoter and regulatorysegments necessary for the expression of that protein or peptide in thatcell.

For example, in one preferred embodiment of the present invention, thehelper cell includes a set of RNAs which include (a) a replicon RNAincluding RNA encoding an alphavirus packaging sequence and an insertedheterologous RNA, (b) a first helper RNA including RNA encoding thealphavirus E1 glycoprotein and the alphavirus E2 glycoprotein, and (c) asecond helper RNA including RNA encoding the alphavirus capsid proteinso that the alphavirus E1 glycoprotein, the alphavirus E2 glycoproteinand the capsid protein assemble together into alphavirus particles inthe host cell.

In an alternate embodiment, the replicon RNA and the first helper RNAare on separate molecules, and the replicon RNA and the second helperRNA are on a single molecule together, such that a first molecule, i.e.,the first helper RNA, including RNA encoding at least one but not all ofthe required alphavirus structural proteins, and a second molecule,i.e., the replicon RNA and second helper RNA, including RNA encoding thepackaging segment, the inserted heterologous DNA and the capsid protein.Thus, the capsid protein is encoded by the second helper RNA, but thesecond helper RNA is located on the second-molecule together with thereplicon RNA. For example, in one preferred embodiment of the presentinvention, the helper cell includes a set of RNAs including (a) areplicon RNA including RNA encoding an alphavirus packaging sequence, aninserted heterologous RNA, and an alphavirus capsid protein, and (b) afirst helper RNA including RNA encoding the alphavirus E1 glycoproteinand the alphavirus E2 glycoprotein so that the alphavirus E1glycoprotein, the alphavirus E2 glycoprotein and the capsid proteinassemble together into alphavirus particles in the host cell.

In one embodiment of the present invention, the RNA encoding thealphavirus structural proteins, i.e., the capsid, E1 glycoprotein and E2glycoprotein, contains at least one attenuating mutation. The phrases“attenuating mutation” and “attenuating amino acid,” as used herein,mean a nucleotide mutation or an amino acid coded for in view of such amutation which result in a decreased probability of causing disease inits host (i.e., a loss of virulence), in accordance with standardterminology in the art, See, e.g., B. Davis, et al., Microbiology 132(3d ed. 1980), whether the mutation be a substitution mutation or anin-frame deletion mutation. The phrase “attenuating mutation” excludesmutations which would be lethal to the virus. Thus, according to thisembodiment, at least one of the first helper RNA and the second helperRNA includes at least one attenuating mutation. In a more preferredembodiment, at least one of the first helper RNA and the second helperRNA includes at least two, or multiple, attenuating mutations. Themultiple attenuating mutations may be positioned in either the firsthelper RNA or in the second helper RNA, or they may be distributedrandomly with one or more attenuating mutations being positioned in thefirst helper RNA and one or more attenuating mutations positioned in thesecond helper RNA. Appropriate attenuating mutations will be dependentupon the alphavirus used. For example, when the alphavirus is VEE,suitable attenuating mutations may be selected from the group consistingof codons at E2 amino acid position 76 which specify an attenuatingamino acid, preferably lysine, arginine, or histidine as E2 amino acid76; codons at E2 amino acid position 120 which specify an attenuatingamino acid, preferably lysine as E2 amino acid 120; codons at E2 aminoacid position 209 which specify an attenuating amino acid, preferablylysine, arginine, or histidine as E2 amino acid 209; codons at E1 aminoacid 272 which specify an attenuating mutation, preferably threonine orserine as E1 amino acid 272; codons at E1 amino acid 81 which specify anattenuating mutation, preferably isoleucine or leucine as E1 amino acid81; and codons at E1 amino acid 253 which specify an attenuatingmutation, preferably serine or threoinine as E1 amino acid 253.

In an alternate embodiment, wherein the alphavirus is the South AfricanArbovirus No. 86 (S.A.AR86), suitable attenuating mutations may beselected from the group consisting of codons at nsP1 amino acid position538 which specify an attenuating amino acid, preferably isoleucine asnsP1 amino acid 538; codons at E2 amino acid position 304 which specifyan attenuating amino acid, preferably threonine as E2 amino acid 304;codons at E2 amino acid position 314 which specify an attenuating aminoacid, preferably lysine as E2 amino acid 314; codons at E2 amino acidposition 376 which specify an attenuating amino acid, preferably alanineas E2 amino acid 376; codons at E2 amino acid position 372 which specifyan attenuating amino acid, preferably leucine as E2 amino acid 372;codons at nsP2 amino acid position 96 which specify an attenuating aminoacid, preferably glycine as nsP2 amino acid 96; and codons at nsP2 aminoacid position 372 which specify an attenuating amino acid, preferablyvaline as nsP2 amino acid 372. Suitable attenuating mutations useful inembodiments wherein other alphaviruses are employed are known to thoseskilled in the art. Attenuating mutations may be introduced into the RNAby performing site-directed mutagenesis on the cDNA which encodes theRNA, in accordance with known procedures. See, Kunkel, Proc. Natl. Acad.Sci. USA 82:488 (1985). Alternatively, mutations may be introduced intothe RNA by replacement of homologous restriction fragments in the cDNAwhich encodes for the RNA, in accordance with known procedures.

A protective molecule in one embodiment may be a nucleic acid moleculeof interest also referred to as a gene of interest and refers to anucleic acid molecule which may or may not represent an entire gene ofthe microorganism. As used herein, the terms nucleic acid orpolynucleotide refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The nucleicacid molecule used to make the vaccine may be the same sequence obtainedfrom the sample, or can refer to a sequence synthetically produced basedupon the sequence obtained from the sample. As such, the terms includeRNA and DNA, which can be a gene or a portion thereof, a cDNA, asynthetic polydeoxyribonucleic acid sequence, or the like, and can besingle-stranded or double-stranded, as well as a DNA/RNA hybrid.Furthermore, the terms are used herein to include naturally-occurringnucleic acid molecules, which can be isolated from a cell, as well assynthetic molecules, which can be prepared, for example, by methods ofchemical synthesis or by enzymatic methods such as by the polymerasechain reaction (PCR). Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes8:91-98). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The protective molecule may also be an RNA interfering molecule or onefrom which an RNA interfering molecule is produced, or which encodes apolypeptide or fragment thereof that produces a protective and/or immuneresponse in the animal when administered to the animal. A noted above,it is at times also referred to as a protective molecule or protectiveantigen determinant. In certain embodiments where a polypeptide isprovided, the polypeptide may be at least two, three, four, five, six,seven, eight, nine or ten or more amino acids or more. A peptide isgenerally considered to be more than fifty amino acids. The terms“fragment,” “derivative” and “homologue” when referring to thepolypeptides according to the present invention, means a polypeptidewhich retains essentially the same biological function or activity assaid polypeptide, that is, act as an antigen and/or provide treatmentfor and/or protection against disease. Such fragments, derivatives andhomologues can be chosen based on the ability to retain one or more ofthe biological activities of the polypeptide, that is, act as aprotective agent and/or provide treatment for and/or protection againstthe pathogen. Thus, a homologue includes a polypeptide from a differentstrain or genus that retains essentially the same biological function oractivity as the polypeptide. The polypeptide vaccines of the presentinvention may be recombinant polypeptides, natural polypeptides orsynthetic polypeptides, preferably recombinant polypeptides.

A protective molecule, then may be the nucleic acid molecule of interestobtained from the biological sample, whether obtained directly orproduced (as synthetically) from the nucleic acid molecule of interest;the polypeptide it produces; interfering RNA such as antisense or dsRNA;DNA producing the antisense or dsRNA; the replicon particles comprisingthe nucleic acid molecule, antisense or dsRNA or a combination whichcomprises the vaccine. The animal may or may not produce antibodies inresponse, but the animal will have decreased morbidity or mortalityresulting from administration of the vaccine, and as described furtherherein.

The invention can be applied to any microorganism/pathogen that causesadverse impact in a aquatic invertebrate and is not limited to anyparticular such microorganism. In particular, the present inventionprovides for methods to immunize against, or to prevent or to reduce thesymptoms caused by, infection of such an animal with a pathogenicorganism. Thus when referring to a microorganism it is meant to includeany such disease-causing agent, for example, a virus, bacteria, fungus,or protozoan parasite. Protection from disease is provided by thevaccine of the invention, that is, protection from all or some of theadverse impact on the animal's health.

Without intending to be limiting, examples of disease causing agents inaquatic invertebrates include Picornavirales viruses such asMarnavirdae, and Dicistronvirdae, Calciviridae such as San Miguel sealion viruses (SMSV) which can infect invertebrates, Nodaviridae such asPenaeus vannamei Nodaviridae (PvNV) affecting shrimp, Iridovirusaffecting shrimp and prawns, Ronivirdae such as Yellow Head Virus (YHV)affecting shrimp, prawns and krill, Bunyaviridae viruses, such asMourilyan virus (MoV) impacting shrimp, Birnaviridae (such as InfectiousPancreatic Necrosis Virus (IPNV) which can impact oysters), icosahedralvirus causing Oyster velar virus disease (OVVD), Nocardia sp. bacteriuminfecting oysters and causing nocardiosis, Vibrio angudarum, V.alginolyticus and V. tubiashii infecting bivalves, Aeromonas hydrophilainfecting snails, Rickettsiae, Chlamydiae and Mycoplasmis infectingbivalve molluscs, Leucothrix mucor infecting clams, Haliphthorosmilfordensis infecting oyster drill, Leptolegnia or Leptoleginellamarina infecting clams, Perkinsus marinus meront and Perkinsusatlanticus infecting molluscs, Haplosporidium sp., Bonamia sp. andMinchinia sp. infecting molluscs, Thraustochytrid infecting squid,nudibranch and octopus, and Perkinsus quqwadi affecting scallop.

Without intending to be limiting, examples of such disease causingagents in shrimp include White Spot Syndrome Virus (WSSV) Taura SyndromeVirus (TSV), Infectious Myonecrosis Virus (IMNV), Infectious hypodermaland hematopoietic necrosis virus (IHHNV), or Penaeus stylirostrisdensovirus (PstDV), Baculovirus of penaeid shrimp (BP), Rhabdovirus ofpenaeid shrimp (RPS), Gill-associated virus (GAV), Yellow head virus(YHV), Lymphoid organ-associated virus (LOVV), Lymphoidal parvolikeviral disease (LPV), hepatopan-creatic parvo-like virus (HPV), orPenaeus monodon densovirus (PMDV) Baculoviral midgut gland necrosisvirus (BMN), Monodon baculovirus (MBV) Reo like virus diseases (REO),Rhabdovirus (RPS), Macrobrachium rosenbergii nodavirus (MrNV),Laem-Singh virus (LSNV), Mourlyan virus (MoV), Vibrio vulnificus Vibroparahaemolyticus, Vibrio anguillarinum, Vibrio penaeicida, NecrotizingHepatopancreatitis Bacterium (NHPB), Vibrio harveyi, Spiroplasma,Mycobacterium, Streptococcus spp., Ciliates, Gregarines, ParasiticHelminths, Fusarium spp., Microsporidia, and Haplosporidia.

Also of importance in protecting aquatic animals, and in particularshrimp and other farmed aquatic invertebrate animals from disease israpid production of a vaccine for the disease. The use of the RepliconParticle technology described here in the invention allows for such arapid response by introducing into the Replicon Particle the gene ofinterest to efficiently and quickly produce a vaccine. (Vander Veen etal. Rapid development of an efficacious swine vaccine for novel H1N1.PLoS Cum 2009 Oct. 29; 1:RRN1123.)

The invention is particularly useful in protecting shrimp from disease,as there is a need for vaccines for shrimp, in particular farmed shrimp.One such disease, for example, is White Spot Syndrome Virus (WSSV).Current production practices focus on pathogen exclusion by stockingspecific pathogen free (SPF) larvae, decontamination and filtration ofwater to prevent pathogen introduction, and strict biosecurity at thehatchery pond sites. These can be effective as long as virus remainsexcluded, but this is extremely challenging due to the prevalence ofWSSV in estuarine waters in shrimp producing areas. It also continues tocause devastating financial losses due to such acute mortality in naivesusceptible SPF populations.

Several control strategies have been utilized experimentally that showsome degree of promise. Fenneropenaeus indicus (Indian Prawn) was shownto be protected by the oral administration of formalin inactivated WSSVvirions. (Singh, I. S. B., Manjusha, M., Pai, S. S., Philip, R., 2005.Fenneropenaeus indicus is protected from WSSV-induced disease by oraladministration of inactivated WSSV. Disease of Aquatic Organisms 66,265-270.) However, this effect seems to be variable and no protectionwas conferred to Litopenaus vannamei (Pacific White Shrimp), the primaryaquaculture species, when orally administered (Loy and Harrisunpublished). No significant differences in mortality or quantitativeReal Time PCR viral genome copy number were observed between vaccinatesand controls in a homologous challenge study (Loy and Harrisunpublished).

Protein subunit vaccines to WSSV envelope proteins have been shown to beeffective at conferring protection to WSSV infection in both shrimp andcrayfish. WSSV contains 4 major envelope proteins with no known homologyto other virus proteins; these include VP28, VP26, VP24, and VP19. VP28is present on the outer membrane and is involved in cellular entry.(McClennen, C. White Spot Syndrome Virus, The Economic, Environmentaland Technical Implications on the Development of Latin American ShrimpFarming. Master of Arts in Law and Diplomacy Thesis. 2004, In Press.)VP28 antisera has been shown to neutralize virus in vivo (McClennen,supra.) Recent studies have demonstrated that these four major envelopeproteins bind to form a complex via several pairwise proteininteractions and one self-association (VP28). (Zhou, Q., Xu, L., Li, H.,Qi, Y.-P., Yang, F., 2009. Four Major Envelope Proteins of White SpotSyndrome Virus Bind To Form A Complex. Journal of Virology 83,4709-4712.)

Subunit vaccines consisting of both VP28 and VP19 conferred protectionto WSSV infection, however protection was short-lived such that 25 daysfollowing administration no protection was conferred. (Witteveldt, J.,Vlak, J. M., Van Hulten, M. C. W., 2004. Protection of Penaeus monodonagainst white spot syndrome virus using a WSSV subunit vaccine. Fish &Shellfish Immunology 16, 571-579.) DNA vaccines to various WSSV envelopeproteins have also demonstrated some utility in preventing infection.Naked DNA vaccines for VP28 and VP281 were injected into Penaeus monodondemonstrable-protection was observed for up to 7 weeks. (Rout, N.,Kumar, S., Jaganmohan, S., Murugan, V., 2007. DNA vaccines encodingviral envelope proteins confer protective immunity against WSSV in blacktiger shrimp. Vaccine 25, 2778-2786.) However, injection of naked DNA toindividuals is not ideal in a commercial setting due to cost ofproduction and feasibility of individual animal injection in the field.Ning et al. demonstrated that Salmonella typhimurium expressing VP28conferred protection against infection for up to 25 days following oraladministration. However, the bacteria remained in the crayfish for onlyseven days. Fu et al. showed that Bacillus subtilis spores andvegetative cells expressing VP28 protected Procambarus clarkii(Freshwater crayfish) against WSSV infection 14 days afteradministration, and that crayfish treated with spores had a highersurvivability than those given vegetative cells. However, problems withusing attenuated bacteria expression systems exist. They still requirethe introduction of live organisms that have the ability to revert tovirulence and may be pathogenic to humans. Due to current problems withall the existing techniques in vaccinating for white spot virus, wepropose a different platform for vaccination.

This body of previously published work focused on either dsRNA or usingprotein delivery as a vaccine or receptor “blocker” for these viraldisease. Here, in one embodiment, a vector is used to deliver singlestranded RNA that is the sequence complement of specific viralsequences, and upon infection creates a RNA/RNA complementary structure.An embodiment involves delivering this RNA or using a nucleic acidmolecule of the pathogen using an alphavirus derived replicon particlevector. Another embodiment uses dsRNA alone or in a replicon particlevector.

Infectious myonecrosis virus (IMNV) is another problematic disease inshrimp and is a non-enveloped, small (40 nm) icosohedral, monosegmented,dsRNA virus, and is a member of the Totiviridae. (Poulos, B. T., Tang,K. F. J., Pantoja, C. R., Bonami, J. R., Lightner, D. V., 2006.Purification and characterization of infectious myonecrosis virus ofpenaeid shrimp. Journal of General Virology 87, 987-996.) This diseasewas subsequently reproduced in specific pathogen free (SPF) animals byinjection of sucrose density gradient purified virions fulfillingRivers' postulates. (Poulos et al., 2006, supra.) IMNV disease ischaracterized by skeletal muscle necrosis in the distal abdominalsegments followed by mortality, especially following periods of acutestress. Histopathologically, animals demonstrate a characteristiccoagulative necrosis of skeletal muscle with fluid accumulation inbetween muscle fibers, along with pronounced hypertrophy of the lymphoidorgan due to accumulation of spheroids. (Poulos et al., 2006, supra.)IMNV was first discovered in 2003 after a severe outbreak in NE Brazilin 2002 of high mortalities and animals exhibiting necrosis in the tailmuscle. It is estimated that in 2003 alone IMNV cost Brazil millions ofdollars in losses. (Lightner, D. V., 1999. The Penaeid Shrimp VirusesTSV, IHHNV, WSSV, and YHV: Current Status in the Americas, AvailableDiagnostic Methods, and Management Strategies. Journal of AppliedAquaculture 9, 27-52.) IMNV was recently confirmed in Indonesia in 2006,and presents a very real risk for spread throughout the world. As thereare no current therapies or vaccines, and little is known about theepidemiology of this virus, this would cause significant impact on thecommercial shrimp farming industry. (Senapin, S., Phewsaiya, K., Briggs,M., Flegel, T. W., 2006. Outbreaks of infectious myonecrosis virus(IMNV) in Indonesia were confirmed by genome sequencing and use of analternative RT-PCR detection method. Aquaculture 266, 32-38.) Inprevious outbreaks, mortalities ranged from 40% to 70% with largeimpacts in production even following large reductions in stockingdensities. Feed conversion ratio (FCR) varied from a normal 1.5 toupwards of 4.4. (Andrade, T. P. D., Srisuvan Thinnarat, Tang, K. F. J.,Lightner, D. V., 2007. Real-time reverse transcription polymerase chainreaction assay using TaqMan probe for detection and quantification ofInfectious myonecrosis virus (IMNV). Aquaculture 264, 9-15.) This hasbeen associated with seasonal fluctuations during the dry season, andthe most significant factors associated with IMNV outbreaks were longrearing periods and high stocking densities. (Arms da Silva, V., dosSantos, F. L., Bezarro, S. S., Pedrosa, V. F., Mendes, P., Mendes E. S.,2010, A multi-season survey for infectious myonecrosis in farmed shrimp,Litopenaeus vannamei, in Pernambuco, Brazil. Journal of InvertebratePathology 104, 161-165.) Due to the current and tremendous futurepotential impact this disease has on the shrimp industry, development ofa vaccine or mitigation strategy is prudent. The aim of this work was todiscover RNAi trigger sequences that would elucidate a protectiveresponse when inoculated into animals at a low dose and for extendedperiods of time following inoculation.

The IMNV genome contains two open reading frames (ORFs). The IMNV genomeis disclosed in Nibert et al, (2007) Journal of General Virology88:1315-1318. and at GenBank accession Number AY570982 by Poulos et al.(2006) and also at Genbank accession Number EF061744 by Senapin et al.(2007). The two GenBank sequences differ by one nucleotide, where thePoulos et al. sequence is 7560 by and the Senapin et al. sequence has anadditional nucleotide insertion of adenine and is 7561 by in length. Thesequence of Poulos et al. is shown in SEQ ID NO: 66 and of Senapin atSEQ ID NO: 67. The sequence of Senapin contains a single nucleotideinsertion of adenine at by 7431 of the genomic sequence. The polypeptideencoded by the ORF1 sequence of Poulous et al. sequence is shown at SEQID NO: 68, and the polypeptide encoded by the ORF1 sequence of Senapinis shown at SEQ ID NO: 69. The polypeptide encoded by encoded by thePoulous et al. ORF2 sequence is shown at SEQ ID NO: 70 (GenBank No.AAT67231.1) and that encoded by the Senapin et al. sequence is shown atSEQ ID NO: 71 (GenBank ABN05325.1). This area of difference was nottargeted in the work here. The ORF1 encodes the major capsid protein(nucleotides 136-4953 of SEQ ID NO: 66 or 67 and identified as SEQ IDNO: 72) and ORF2 (nucleotides 5241-7451, SEQ ID NO: 73) encodes a 736amino acid RNA dependent RNA polymerase (RdRp) (Poulos et al., 2006,supra; see also Nibert, 2007, supra.) ORF1 encodes a 179 kDa protein(1605 amino acids) including the N-terminal sequence of the major capsidprotein. The capsid is isometric with a diameter of approximately 400angstroms. Recent studies of the IMNV genome have revealed a “2A-like”cleavage and “shifty heptamer” that may contribute to a capsidprotein-RdRp fusion protein as well as three cleavage proteins of ORF 1.These have been described as “Peptide 1,” “Peptide 2,” and “Peptide 3.”There remains some speculation as to the role of these proteins.“Peptide 1” spanning bases 136-415 (SEQ ID NO: 74, is a 10 kDa, 93 aminoacid region at the N-terminus of ORF 1, shares sequence similaritieswith known dsRNA binding proteins, and may be involved in host immunesuppression. (Tang et al., 2008 Infectious myonecrosis virus has atotivirus-like 120-subunit capsid, but with fiber complexes at thefivefold axes. PNAS 105:17526-17531.) “Pep2” a 32 kDa, 284 aa product,spanning bases 415-1266 (SEQ ID NO: 75) and “Pep3” a 38 kDa, 327 aaproduct, spanning 1267-2247 (SEQ ID NO: 76), together represent thefirst 704 amino acids of ORF1, have been speculated to be candidateminor proteins visualized on denaturing gels, however this remainsspeculative in nature (Tang, et al., 2008, supra).

Three target regions, spanning the length of the viral genome, wereselected as initial targets for dsRNA generation (see FIG. 2). The firstwere sequences corresponding to the N-terminal region of ORF1 frame 1,predicted to contain two co-translationally cleaved products, spanningPeptide 1 and 2 (dsRNA95-475, here SEQ ID NO: 1). In addition portionsof the major capsid protein (MCP) (dsRNA3764-4805, here SEQ ID NO: 4)and RNA dependent RNA polymerase (RdRp) dsRNA5518-6391 (here SEQ ID NO:5) were selected for dsRNA generation. Peptide 1 (93 aa) and 2 (284 aa)are encoded by nucleotides 136-415 (SEQ ID NO: 74) and 415-1266 (SEQ IDNO: 75) of the IMNV genome within ORF 1 frame 1, respectively, and theirfunctions remain uncharacterized. Peptide 1 shares sequence homologywith previously described dsRNA binding proteins The major capsidprotein (909 amino acids) of IMNV is encoded by nucleotides 2227-4953(SEQ ID NO: 77) within ORF 1, frame 1. The RNA dependent RNA polymerase(736 amino acids) is encoded by nucleotides 5241-7451 (SEQ ID NO: 78)within ORF2, frame 3. A non-specific control dsRNA was designed to anexogenous sequence corresponding to enhanced green fluorescent protein(eGFP). Shorter dsRNAs were designed within the area of the IMNV genomethat encodes Peptide 1 (FIG. 2). Two dsRNAs were generated as 100 bytruncations from the 5′ (bp193-475, SEQ ID NO: 2) or 3′ (bp95-376, SEQID NO: 3) end of the original dsRNA95-475 (SEQ ID NO: 3).

The means and methods of producing such a vaccine are known to oneskilled in the art and many variations and approaches to such productionare known and expected to be further developed. The following sets forthas examples some of the many options available to produce and administersuch a vaccine. A discussion of an example of various means forproducing and administering vaccines of the invention is described atHarris et al., U.S. Pat. No. 7,622,254, incorporated herein by referencein its entirety.

One means of producing such a vaccine is to produce an autogenousvaccine, that is, to use a a method of producing a vaccine is providedwhich protects the animal from adverse effects of a biotype of apathogenic microorganism. Disclosed here and at US patent application“Method of rapidly producing improved vaccines for animals” U.S. Ser.No. ______, and which claims priority to U.S. provisional applicationU.S. Ser. No. 61/407,297 filed Oct. 27, 2010, is a description ofproducing a vaccine from an autogenous source. The contents of each areincorporated herein by reference in their entirely. Also described andas shown in the examples below is an autogenous source of a nucleic acidmolecule of interest and protective molecule. In the autogenous process,in summary, a biological sample is obtained from an animal which hasbeen exposed to a microorganism, a nucleic acid molecule of interest offragment thereof of the microorganism is obtained from the sample and aprotective molecule produced from the nucleic acid of interest. Theprotective molecule may be a nucleic acid molecule comprising thesequence or a fragment of the nucleic acid molecule of interest, may bea polypeptide or fragment produced by the nucleic acid molecule ofinterest, or may be an RNA molecule that is antisense to the nucleicacid molecule of interest or forms a dsRNA that corresponds to all or aportion of the nucleic acid molecule of interest or nucleic acidmolecule producing same or replicon particle comprising or producingsame. With such a process, an improved vaccine is provided to protectanimals against a pathogen which can be prepared very rapidly and whichaddresses the problem of providing protection to animals against a newor evolving biotypes. The vaccine is an autogenous vaccine, that is, itis prepared from the nucleic acid molecule from an infectious agentpresent on a specific farm, flock, herd, pond or geographic region. Itis not necessary to isolate the infectious agent in the laboratory toobtain the gene. It is prepared from the nucleic acid ofmicroorganism(s) present in an animal or a group of animals which havebeen exposed to a biotype of a pathogenic microorganism. Such animalsfrom which the nucleic acid molecule is obtained are those living in anenvironment such that one can expect are likely to have been exposed tothe same pathogen biotype. By way of example, without limitation, wheresuch animals are livestock animals, they may be found living in a ranch,feed yard, farm, pond or region and with sufficient contact such thatone skilled in the art would expect such animals to have come intocontact or are likely to come into contact with the same pathogen. Uponpreparation of the autogenous vaccine, these animals would then bevaccinated with the vaccine. As provided for with by the AmericanVeterinary Medical Association (AVMA), adjacent or non-adjacent groupsof animals considered to be at risk may also be vaccinated. Seewww.avma.org/issues/policy/autogenous_biologics.asp “Guidelines for Useof Autogenous Biologics” (Oversight: COBTA; EB approved-1993; reaffirmed11/97; reaffirmed 4/01; revised 3/06, 11/09). When referring to anautogenous vaccine is meant to include this current definition of theAVMA in which animals considered to be at risk may be vaccinated. Also,an individual animal may be the sole animal for which the autogenousvaccine is prepared. The source of the microorganism nucleic acidmolecule of interest is any convenient biological sample such as animaltissue, fluid or cell which are expected to have the nucleic acid of themicroorganism present, whether blood, skin, organ tissue, body fluids orthe like.

The term biotype refers to distinguishing a pathogenic agent by one ormore characteristics over other members of the pathogenic species. Theinvention is particularly useful in providing a process to quicklyproduce a vaccine that is useful with different and/or new biotypes of apathogen, and in an embodiment is especially useful where a biotype isfound in a particular group of exposed animals or with potential forexposure to that biotype. Using current methods, a vaccine that isavailable may not be helpful against a different or newly evolvingbiotype. This invention provides a process where a vaccine that isuseful with the new or different biotype is quickly developed. A biotypevariant of a species can be distinguished by a variety of one or morecharacteristics, such as ribosomal RNA sequence variation, DNApolymorphisms, pathogenic response, response of the exposed animal to aspecific vaccine, serological typing of toxin production or many otherpossible variations depending upon the pathogenic agent (see e.g.,Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edit., ColdSpring Harbor Laboratory, cold Spring Harbor, N.Y. 2001; DNA cloning APractical Approach, Vol. I and II, Glove, D. M. edit. IRL Press Ltd.,Oxford, 1985; Harlow and Lane, Antibodies a Laboratory Manual, ColdSpring Harbor Publications, N.Y. 1988). By way of example, withoutlimitation, influenza can be biotyped by distinguishing it by subtypeand cluster. The category is determined by differences of their internalproteins, and further by differences of the hemagglutinin andneuraminidase proteins. A hemagglutinin inhibition test can be used, inone example, where a sample of specified dilution is applied to redblood cells and the titer determined by the maximum dilution thatproduces agglutination. Antibodies to the virus prevent attachment tored blood cells and thus hemagglutination is inhibited when antibodiesto influenza virus are present. Results are reported as the reciprocalof maximum dilution that provides visible agglutination. See. e.g., Katzet al. (2009) Morbid. Mortal. Weekly Rep. 58 (19) 521-524.

Another example of biotyping is glycan typing where genotypes aregrouped based on their glycosylation patterns. Such a process isdescribed at Harris et al., U.S. Pat. No. 7,622,254, incorporated hereinby reference in its entirety (see especially, for example, table 7columns 48 and 49). For example, the strains of PRRSV (PorcineReproductive Respiratory Virus) are classified based on whether they areEuropean or North American strains. In another aspect of typing thePRRSV strains, the first letter is either EU (European like) or NA(North American like) to designate the genotype cluster. EU refers toisotypes of PRRSV characterized by conserved glycans at position 46, 53,or both in GP5. As used herein, NA refers to isotypes of PRRSVcharacterized by conserved glycans at position 44, 51, or both in GP5.Each strain is given a number corresponding to the number ofglycosylation sites located in the ectodomain of GP5 amino acid sequenceshown in Table 7 of '254, but excludes highly conserved glycans locatedat aa44 and 51 for NA strains and aa46 and 53 for EU strains. Thus, NA-0refers to the ectodomain of GP5 of NA strain that has no glycan and EU-0refers to the ectodomain of GP5 of an EU strain that has no glycan. Forexample, NA-1 refers to the ectodomain of GP5 of a North American strainthat has 1 glycan located on the ectodomain of GP5 excluding highlyconserved glycans located at aa44 and 51 for NA strains. Table 1represents such a glycantyping of PRRSV.

TABLE 1 PRRSV Glycantype^(a) Number of predicted glycans^(b,c) NA-0 0^(d) NA-1 1 NA-2 2 NA-3 3 NA-4 4 NA-n n EU-0 0 EU-1 1 EU-2 2 EU-3 3EU-4 4 EU-n n ^(a)NA + North American, EU = European ^(b)Number ofglycans located on the ectodomain of GP5 excluding highly conservedglycans located at aa44 and 51 for NA strains and aa46 and 53 for EUstrains. When these glycans are absent they should be noted as follows:if an NA-1 strain lacks a glycan at aa44 it is described as NA-1 (Δ44).^(c)As the number of predicted glycans increases so does the resistanceto inducing protective (neutralizing) antibodies and/or susceptibilityto such antibodies. NA-0 and EU-0 are predicted to be the parent strainsfor all NA and EU strains respectively. Thus these viruses should beincluded in attempts to generate cross-reacting antibodies. After NA-0and EU-0, glycantyping may be a predictor of heterology which iscurrently poorly defined for PRRSV.

Any biotyping method to distinguish a pathogen from another of thespecies may be used in the invention.

This provides a useful approach to the generation of autogenousvaccines. In current processes used, the whole organism is isolated,then attenuated or killed, and the animal vaccinated with the preparedvirus. By way of example, U.S. Pat. No. 4,692,412 to Livingston et al.describes a method for preparing an autogenous vaccine for neoplasticdiseases by mixing a sterile blood sample containing Progenitorcryptocides with sterile distilled water, incubating the admixtures,killing or inactivating the Progenitor cryptocides in the admixtures,microfiltering the admixture to remove blood cells and diluting thefilature to form the vaccine. Here, rather than use the whole organism(either live or inactivated) as the vaccine, one uses only one or moremicroorganism individual gene(s) of interest (GOI) also referred to asthe nucleic acid molecule of interest (NOI) or fragments thereof,derived from the pathogen and/or the protein such gene(s) encode thatmakes up the autogenous vaccine. Surprisingly, it is possible to producea vaccine using such nucleic acid molecules and to provide an effectivevaccine which protects the animal. The gene of interest refers to anucleic acid molecule which may or may not represent an entire gene andmay be one from which an RNA interfering molecule is produced, orencodes a polypeptide or fragment thereof that produces a protectiveand/or immune response in the animal when administered to the animal. Asone skilled in the art appreciates, the actual vaccine uses a protectivemolecule and may contain the gene of interest or fragment thereof, ormay contain the polypeptide or fragment thereof producing the protectiveresponse, or may contain the interfering RNA or may contain acombination. The process is advantageous for the following reasons:

1) The new vaccines do not contain live virus. Current modified livevirus (MLV) vaccines could not be used in an eradication effort due totheir ability to spread, revert to virulence, or recombine with fieldstrains.

2) The new vaccines can differentiate infected from vaccinated animals.Current MLV and killed vaccines use the whole virus. Here the entireinfective agent is not included.

3) Autogenous vaccines and can be produced quickly. Autogenous vaccinesare desirable for animal disease treatment due to strain/biotypevariation and lack of cross-protection. ARP vaccines can be producedfaster (1 month and even less) than traditional autogenous vaccines (3months) allowing for a quicker response.

In a yet further embodiment, one can optionally first determine ifpreparation of a new autogenous vaccine as described is more advisableby determining the antigenic drift of the pathogen. In such anembodiment, one obtains a sample comprising the microorganism asdescribed above, and may optionally determine what biotype it is. Usinginfluenza as an example, the subtype and cluster may be determined. Ingeneral, influenza viruses are made up of an internal ribonucleoproteincore containing a segmented single-stranded RNA genome and an outerlipoprotein envelope lined by a matrix protein. The genome of influenzaviruses is composed of eight segments of linear (−) strand ribonucleicacid (RNA), encoding the immunogenic hemagglutinin (HA) andneuraminidase (NA) proteins, and six internal core polypeptides: thenucleocapsid nucleoprotein (NP); matrix proteins (M); non-structuralproteins (NS); and three RNA polymerase (PA, PB1, PB2) proteins. Duringreplication, the genomic viral RNA is transcribed into (+) strandmessenger RNA and (−) strand genomic cRNA in the nucleus of the hostcell. Each of the eight genomic segments is packaged intoribonucleoprotein complexes that contain, in addition to the RNA, NP anda polymerase complex (PB1, PB2, and PA). As noted, influenza is groupedinto three categories, based on the absence of serologic crossreactivity between their internal proteins: influenza A, B and C.Influenza A viruses are further classified into groups by antigenicdifferences of hemagglutinin and neuraminidase proteins. Examples ofsubtypes and further classification into clusters are shown in Table 2below. The hemagglutinin antigens of influenza viruses change frequentlyin antigenic specificity as a result of changes in the HA and NA aminoacid sequence.

TABLE 2 Subtype Representative HA sequence Cluster H3N2A/Swine/Indiana/7688/2007 4 (H3N2) cH1N1 A/swine/Iowa/15/1930(H1N1) αrH1N1 A/swine/Illinois/4661/2009 β (H1N1) H1N1A/Swine/Illinois/11678/2008 γ (H1N2) 2009 A/California/04/2009(H1N1) γH1N1 huH1N1 A/Swine/Illinois/49271/2008 δ huH1N1 (H1N2)

In an embodiment of the invention, the antisera is obtained, anddetermined if it is the same biotype—in the present example if it reactsin the same manner as a standard obtained using existing vaccine. Thiscan be measured in the case of influenza by using an hemagluttinininhibition test as a standard, as described infra. If it reacts thesame, then the existing vaccine can be used, if it does not match, thena new vaccine may be prepared. This is a means of measuring drift, thatis change in the antigenic components. This is more effective thanmeasuring shift, that is a major change to a new subtype. By measuringantigenic components, the virus may still fall within the same cluster,yet demonstrate a sufficient drift that a new vaccine would be moreeffective. While presented by way of example regarding an influenzavirus, clearly one could apply such a process to any microorganism.

For many pathogens the protective molecule(s) needed to induceprotection are known. The gene of interest of a pathogen is firstamplified from a diagnostic sample originating from the farm. While onecan isolate and purify the pathogen, it is not necessary with thismethod. Not only in such an embodiment does this eliminate anunnecessary step and speed the production process, it removes the needto have an isolated pathogen. The gene may be isolated or any protectiveportion of it isolated by using any available method such as PCR. Thegene is then used to prepare the vaccine and ultimately used tovaccinate animals that have been or may be exposed to the pathogen.These vaccines when compared to currently available vaccines would befaster, biotype specific, and compatible with diagnostic tests. Vaccinesso produced and a method of protecting an animal using the vaccine isalso provided.

In one embodiment of the invention, a method of identifying protectivesequences of the virus or nucleic acids that elicit protection isprovided. This method also includes fragments, derivatives, or homologsof the protective molecule. In one aspect, the method comprisesadministering to a test animal such sequences. The test and controlanimals are subsequently challenged with an infectious amount of amicroorganism that causes the disease. Various methods and techniquesfor determining whether protection is provided are known to thoseskilled in the art, including but not limited to, observing a differencebetween the test and control animal in the symptoms of the disease, forexample. A decrease in any of the symptoms observed in the test animalcompared to the control animal indicates that protective molecule(s)provide a degree of protection against disease. Similar symptoms or anincrease in any of the symptoms observed in the test animal compared tothose observed in the control animal indicate that the protectivemolecule(s) do not provide protection.

In another aspect, determining whether the protective molecules providedprotection against infection includes determining the presence orabsence of challenge disease in the test animal by electron microscopyor antibody or assays such as the fluorescent focusing neutralizing(FFN) test or Western blot assay may be used. PCR methods may be used todetermine if the protective molecule is present. Northern blotting candetect the presence of diagnostic sequences. The presence of thechallenge organism indicates that the sequence(s) isolated are noteffective in protecting against the disease and the absence of thechallenge organism indicates that the protective molecule(s) areeffective in protecting against the disease. In another aspect, an ELISAor similar assay is among the types of many varied assays that candetermine if the protective molecule is effective. The ELISA or enzymelinked immunoassay has been known since 1971. In general, antigenssolubilised in a buffer are coated on a plastic surface. When serum isadded, antibodies can attach to the antigen on the solid phase. Thepresence or absence of these antibodies can be demonstrated whenconjugated to an enzyme. Adding the appropriate substrate will detectthe amount of bound conjugate which can be quantified. A common ELISAassay is one which uses biotinylated anti-(protein) polyclonalantibodies and an alkaline phosphatase conjugate. For example, an ELISAused for quantitative determination of laccase levels can be an antibodysandwich assay, which utilizes polyclonal rabbit antibodies obtainedcommercially. The antibody is conjugated to alkaline phosphatases fordetection. In another example, an ELISA assay to detect trypsin ortrypsinogen uses biotinylated anti-trypsin or anti-trypsinogenpolyclonal antibodies and a streptavidin-alkaline phosphatase conjugate.

Clearly, many such methods are available to one skilled in the art toascertain if the protective molecule provides protection, and providesprotection at the levels administered to the animal.

The present inventors also contemplate that the isolated sequences fromthe microorganism of the present invention may be delivered usingvarious vectors and viruses. In an optional embodiment it is possible toprovide an adjuvant in the vaccine. Adjuvants enhance the immunogenicityof an antigen but are not necessarily immunogenic themselves. Adjuvantsmay act by retaining the antigen locally near the site of administrationto produce a depot effect facilitating a slow, sustained release ofantigen to cells of the immune system. Adjuvants can also attract cellsof the immune system to an antigen depot and stimulate such cells toelicit immune responses. Immunostimulatory agents or adjuvants have beenused for many years to improve the host immune responses to, forexample, vaccines. The vaccines of the present invention may be used inconjunction with an adjuvants, for example, lipopolysaccharides,aluminum hydroxide and aluminum phosphate (alum), saponins complexed tomembrane protein antigens (immune stimulating complexes), pluronicpolymers with mineral oil, killed mycobacteria in mineral oil, Freund'scomplete adjuvant, bacterial products, such as muramyl dipeptide (MDP)and lipopolysaccharide (LPS), as well as lipid A, and liposomes.Desirable characteristics of ideal adjuvants may include: (1) lack oftoxicity; (2) ability to stimulate a long-lasting immune response; (3)simplicity of manufacture and stability in long-term storage; (4)ability to elicit both CMI and HIR to antigens administered by variousroutes; (5) synergy with other adjuvants; (6) capability of selectivelyinteracting with populations of antigen presenting cells (APC); (7)ability to specifically elicit appropriate T-cell helper 1 (TH 1) or TH2 cell-specific immune responses; and (8) ability to selectivelyincrease appropriate antibody isotype levels (for example, IgA) againstantigens. An adjuvant used with the present invention need not possessall these characteristics to be used with the present invention.

Another adjuvant which may be used is E. coli heat labile enterotoxin(LT). LT has been used to assist in preventing E. coli induced diarrhea(See for example Limjuco et al., U.S. Pat. Nos. 4,285,931 and4,220,584). However, since its early isolation it has been found thatits use of LT is greatly limited due to toxicity, and is avoided as anadjuvant unless modified in some manner to reduce toxicity. See Zhang etal. (2009) Vet. Rex. Commun. DOI 10/1007/s11259-009-9222-7. Thus therehas been effort to avoid the toxicity problem by changing the sequenceof the enterotoxin or by truncation. See, e.g., Piazza et al., U.S. Pat.No. 7,291,588. However, it is possible to use a non-mutated LT as anadjuvant without toxicity. Such non-mutated LT is that which is nottruncated or otherwise mutated. As a result, an adjuvant non-toxicimpact is provided, and at reduced cost in its manufacture.

The vaccine composition may be introduced into an animal, with aphysiologically acceptable vehicle and/or adjuvant. Useful vehicles arewell known in the art, and include, e.g., water, buffered water, saline,glycine, hyaluronic acid and the like. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being rehydrated prior to administration, as mentionedabove. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, and the like. Inone embodiment of the invention, the protective molecule is encapsulatedsuch that the resulting composition is water resistant. In anembodiment, the molecule is combined with a binder that assists inassociating the molecule with feed, which is particularly useful fororal administration. Such a water resistant binding substance can be anysubstance having such properties. Examples include, without limitation,agarose or other sugar compounds, albumin, alginate or any similarcomposition.

In another embodiment, protective molecules isolated from a particularstrain or biotype can be combined with other sequences and components ofother strains or biotypes or diseases to achieve protection againstmultiple microorganisms. These different microorganism sequences orcomponents may be administered sequentially or progressively oralternately administered simultaneously in an admixture. Sequential orprogressive administration of the vaccine compositions of the inventionmay be required to elicit sufficient levels of immunity to multiplemicroorganism strains. Single or multiple administration of the vaccinecompositions of the invention can be carried out. Multipleadministration may be required to elicit sufficient levels of immunity.Levels of induced immunity can be monitored by measuring amount ofneutralizing secretory and serum antibodies, and dosages adjusted orvaccinations repeated as necessary to maintain desired levels ofprotection.

The protective molecules may be “administered” in any suitable manner,including but not limited to, by immersion in a composition or substancecontaining the protective molecule (as with shrimp, by providing thevaccine in liquid surrounding the shrimp, for example) parenterally, byinjection subcutaneously or intramuscularly, into an organ or cavity ofthe animal, reverse gavage (rectally), and oral, whether per os oringestion of feed, as well as transdermal or by gas exchange. Thevaccine can be administered by any means which includes, but is notlimited to, syringes, nebulizers, misters, needleless injection devices,or microprojectile bombardment gene guns (Biolistic bombardment), via aliposome delivery system, naked delivery system, electroporation,viruses, vectors, viral vectors, or an ingestible delivery systemwherein the protective molecules are consumed, for example, in feed orwater or in any other suitable manner. Oral or immersion administrationprovides advantages in ease of administration and the capacity toadminister the protective molecules to a group of animals. Injection ofthe protective molecules can be useful with brood stock, that is, adultanimals that have reached or are nearing reproductive maturity. Theimmunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, immunogenic and protective.

The quantity to be administered depends on the subject to be treated,including, for example, the capacity of the immune system of theindividual to mount a protective response. Suitable regimes for initialadministration and booster doses are also variable, but may include aninitial administration followed by subsequent administrations. The needto provide an effective amount of the protective molecule will also needto be balanced with cost of providing higher amounts of the protectivemolecule. A cost effective vaccine is one in which the cost of producingit is less than the value one can obtain from using it.

Measurement and determination of efficacy of any of the compositions andvaccines of the invention may be accomplished by any of the many methodsavailable to one skilled in the art. By way of example, withoutlimitation, where the process involves interference with a targetmolecule, target molecule suppression can be measured by quantitativeRT-PCR or RT-PCR or nucleic acid hybridization or Northern blotting ofwhole body or specific tissues RNA extracts, using primer sets thatextend beyond or are completely removed from the region encompassed bythe dsRNA generating primer set.

Where the vaccine involves production of a protein, a straightforwardand quick method can be to perform a Western blot analysis of a samplecandidate vaccine composition to quantitate the amount of polypeptide orfragment thereof in the sample. There are various options available tothe skilled person. In one embodiment, one compares the amount ofpolypeptide to a standard known to be effective with like polypeptidesfrom other biotypes, and either prepares a vaccine where the level ofpolypeptide produced is at least at this standard or higher, or may testthe vaccine with a test animal.

There is uncertainty whether an aquatic invertebrate, such as shrimp,produce antibodies. While an immune defense response has been observed,it is believed that such animals do not produce antibodies as defined bycurrent immunology dogma. (See, Kurtz, Trends in Immunology, Vol. 25 No.5, April 2005.) In the event that measurement of antibody production orlevels of induced immunity can be monitored by measuring antibodyproduction, or in the interest of measuring production of protectivemolecules or the disease agent itself, in order to optimize dosages orvaccinations repeated as necessary to maintain desired levels ofprotection, simple immunoblotting techniques can be used by thoseskilled in the art. For example, an ELISA can be performed. An ELISA canbe performed on a sample collected from an individual vaccinated todetermine whether antibodies to a vaccine comprising the sequence, aderivative, a homologue or a variant or fragment thereof generatedanti-polypeptide antibodies or to determine whether the vaccine moleculewas successfully expressed, using an antibody. The individual's sampleis measured against a reference anti-polypeptide antibody.

The effectiveness of the present vaccine may also be evaluatedquantitatively (for example, a decrease in a measurement of disease ascompared to an appropriate control group) or qualitatively (e.g.,isolation of pathogen or virus from tissue or fluids, and where possibleto detect, detection of antigen in a tissue sample by animmunoperoxidase assay method, etc.). Analysis of symptoms andmeasurement of animal weight gain also demonstated lessening of impactof the disease in the presence of a particular dose. In still anotherexample, ranges of doses may be prepared and protective responsemeasured. A candidate vaccine can be formulated at two or three or moredifferent doses to determine the minimum protective dose. For example,when using RP, in addition to measuring IFA titration, qPCR assays canbe used to determine the number of genome equivalents present in thevaccine and compared to IFA titer to obtain a GE:RP ratio. Such an assayhelps assure uniformity as well. In still another example, such testingindicated that a swine influenza efficacious dose was about 1×10⁸ RP. Byway of another example, without limitation, two or three or more doseranges may be prepared, as in the example below with shrimp andmorbidity and mortality measured upon challenge. The dose selected in apreferred embodiment is that which provides protection to the animalwhich is also cost effective.

Clearly one skilled in the art has many different options available formeasuring effectiveness of the vaccine.

With the present invention, it is possible to achieve protection againstdisease in an aquatic invertebrate, and which protection is provided forlonger periods than have been achieved in such animals. Protectionperiods of more than seven days after at least one challenge or exposureto the pathogenic microorganism have been achieved, and protection of atleast two weeks, at least 20 days, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days or more, havebeen achieved using the invention. The protective response is also shownhere in an embodiment to be specific to the disease as opposed toanother disease, and thus demonstrates specific memory.

The following examples are presented by way of illustration and are notintended to be limiting to the scope of the invention.

Example 1

Here described is one method of producing autogenous replicon particle(ARP also referred to by some practitioners as RNA particle or RepliconParticle) In a first experiment, it was confirmed that RP vaccines werecapable of producing a foreign antigen in vivo that resulted in aninduced immune response in pigs. Pigs were obtained at 2 weeks of age,weighed, tagged, and randomized into 2 groups of 8 pigs each (Table 3).Prior to vaccination, serum was collected to assure pigs were free ofinfluenza antibodies.

TABLE 3 Design of replicon particle (RP) proof of concept study in pigs.Group (n) Treatment (route) Dose Vaccination Schedule 1 (8) RP (IM) (Negcontrol) 10⁸ Day 0 prime, Day 14 boost 2 (8) HA RP (IM) 10⁸ Day 0 prime,Day 14 boost Pigs received either an empty RP or an RP expressing theinfluenza hemagglutinin (HA) protein.

Serum was collected throughout the study for hemagglutination inhibition(HI) assay using the A/Wyoming/03/2003 virus. All pigs were HI negativeprior to starting the study and Group 1 pigs remained negative duringthe study (individual pig data not shown). However a robust and longlasting antibody response to HA protein was induced in Group 2 (Table4). This response started to appear following the priming dose andelevated quickly after the second dose. By Day 21 all pigs had titersbetween 1280 and 5120 and maintained this level through termination ofthe study at 62 days. Although the pigs in this experiment were notchallenged with virulent virus, based on previous studies and generallyaccepted protective level (>40) this level of HI antibody would havebeen protective.

TABLE 4 HI results (inverse titers) of pigs immunized with RP expressingthe influenza HA protein (>40 considered positive). Pigs were immunizedon Day 0 and 14. The trial ended on Day 62. Pig # Day −9 Day 13 Day 21Day 28 Day 41 Day 51 Day 62 4 10 320 2560 2560 2560 2560 1280 8 10 802560 5120 2560 2560 2560 11 10 40 2560 2560 2560 2560 1280 18 10 40 25602560 2560 2560 2560 20 40 20 5120 2560 5120 5120 5120 25 20 80 2560 25605120 5120 5120 26 10 80 2560 5120 2560 2560 2560 30 10 40 5120 2560 25602560 2560

We have successfully shown that RP vaccines can be made that co-expressboth PRRSV GP5 and M proteins and that a heterodimer is formed. Erdman MM, Harris D L, Kamrud K I. Replicon particle co-expression of PRRSV GP5and M proteins. Proc CRWAD 2006. Following successful expression invitro, a pig trial was conducted to determine if the GP5-M RP vaccinewould induce an immune response in pigs. Pigs 2-3 weeks of age wereobtained from a PRRSV negative source. Pigs were tagged, weighed, andrandomized into 2 groups of 8 (Table 5). Serum was collected prior tovaccination to assure pigs had no antibodies to PRRSV.

TABLE 5 Previous PRRSV RP proof of concept study. Group (n) Treatment(route) Dose Vaccination Schedule 1 (8) RP Control (IM) 10⁸ Day 0 prime,Day 14 boost 2 (8) GP5-M RP (IM) 10⁸ Day 0 prime, Day 14 boost Pigs werevaccinated with either an empty RP or an RP co-expressing PRRSV GP5 andM proteins.

All pigs were challenged on Day 48 with a homologous PRRSV strain(HLV013) intramuscular with 2 ml of 10⁶ TCID₅₀. Neither group had virusneutralizing antibodies on Marc 145 cells prior to challenge althoughWestern blotting indicated antibodies were present to the PRRSVproteins. Following challenge, 3 of 8 pigs in the vaccinate group and 0of 8 pigs in the control group had neutralizing titers within 7 days.All pigs were necropsied 14 days after challenge. At necropsy 5 of 8 inthe vaccinate group and 4 of 8 in the control group had neutralizingtiters. However the geometric mean titers (GMT) was higher in thevaccinate group (GMT=27.8) compared to the control group (GMT=9.5).Virus titration of lung lavage indicated that 7 of 8 control pigs werevirus positive and only 4 of 8 vaccinate pigs were virus positive. Lunglesion scoring showed less gross pathology in the vaccinate group (meanscore of 16.3) compared to the control group (mean score of 22.3).

Results and Accomplishments ARP Vaccine Construction

The entire vaccine development process took three weeks from PRRSVpositive serum sample to formulated GP5 and M single promoter ARPvaccines and an additional week to produce the GP5/M double promoter ARPvaccine. Due to the fact that speed of development was important to usin this experiment we opted to conduct the trial by co-injecting the GP5and M single promoter ARP since they took less time to produce and posedless technical challenges. However both the single promoter and doublepromoter approaches are viable options and that both are more rapid thanthe time it generally takes to develop a new conventional autogenousvaccine requiring isolation and growth of the virus.

PCR can be performed on a sample to both provide a positive diagnosis ofPRRS and also generate cDNA of the genes needed to make the ARP vaccine.It is also anticipated that this entire process can be performed in lesstime than it generally takes for a pig producer to get a traditionalautogenous vaccine made.

To develop the new ARP vaccine, a large swine producer that had pigssuffering from clinical PRRSV was identified. Serum samples fromdiseased animals were sent to Iowa State University. RT-PCR was usedboth to confirm diagnosis and produce cDNA of genes targeted for vaccineproduction. Specific primers were used to amplify both the PRRSV GP5 andM genes, ORF 5 and 6 respectively. Although not needed to make thevaccine, the live virus was isolated in order to prepare a traditionalkilled autogenous vaccine and used for challenging the pigs. The isolatewas referred to as PRRSV strain Pennway.

The PRRSV GP5 and M genes were cloned into replicon vectors that arebased on the live attenuated vaccine strain of VEE known as TC-83 (FIG.3). The GP5 and M replicon vectors were analyzed by both IFA and Westernblot to confirm expression of desired proteins. The replicons were thenpackaged into particles via co-electroporation with helper RNAs toproduce ARP. ARP were harvested from culture fluids and the infectioustiter of the ARP preparation was measured by antigen-specific IFA andtested in a CPE assay to assure the absence of replication competentvirus.

Vaccine Formulations

The placebo vaccine consisted of 2 ml of sterile PBS (HyCloneLaboratories).

The ARPs had initial titers of 2.11e9/ml for the GP5 ARP and 3.56e9 forthe M ARP. Each ARP was diluted to a titer of 1e8/ml using PBS. Eachdose consisted of 1 ml of GP5 ARP and 1 ml of M ARP co-injected in thesame syringe.

The inactivated PRRSV vaccine was derived from the same strain used toproduce the ARP vaccine. The virus was grown to 85% CPE on Marc-145cells and harvested from the supernatant. The virus titer was determinedto be 2.7e5 TCID₅₀/ml. The virus was heat inactivated at 65 degrees C.for 90 minutes. A sample of the prep was inoculated onto Marc-145 cellsto confirm the lack of viable virus. The lysate was formulated withEmulsigen-D adjuvant (MVP laboratories, Ralston, Nebr.) by adding 1.6 mlof the lysate with 0.4 ml of adjuvant per dose.

The PRRSV MLV vaccine was Inglevac PRRS ATP (Boehringer IngelheimVetmedica, Inc). The vaccine was used as directed by the manufacturerwhich indicated giving a single 2 ml dose of vaccine.

Pig Trial

Three week old pigs were obtained from a farm in Iowa with no history ofPRRSV infection based on clinical signs and serology. Pigs were eartagged, weighed, and randomized into five groups of ten pigs each inBSL-2 animal facilities at Iowa State University. A description ofgroups is given in Table 5 and a timeline for the vaccination-challengestudy is shown in Table 6. Group 1 contained strict negative controlpigs that were neither vaccinated nor challenged. All pigs in Groups 2,3, and 4 were vaccinated intramuscular (IM) on Day 0 and again on Day28. Group 2 received the placebo vaccine, Group 3 the ARP vaccine, andGroup 4 the inactivated PRRSV vaccine. Group 5 received one dose of theMLV vaccine on Day 0.

TABLE 6 Groups in PRRSV vaccination-challenge study Pigs PRRSV Group (n)Treatment Route challenge 1 10 Strict negatives NA NA 2 10 Placebo IM IN3 10 ARP PRRSV GP5-M IM IN 4 10 Inactivated PRRSV IM IN 5 10 MLV PRRSVIM IN PRRSV = porcine reproductive and respiratory syndrome virus, M =matrix protein, GP5 = glycoprotein 5, ARP = autogenous repliconparticles, MLV = modified live virus NA = not applicable, IM =intramuscular, IN = intranasal

Pigs were challenged IN on Day 56 with 2 ml of 1×10⁵ TCID₅₀/ml ofvirulent PRRSV strain Pennway. Pigs were monitored for clinical signsincluding respiratory distress, lethargy, and recumbancy followingchallenge but none were noted. Pigs were euthanized 21 days afterchallenge, a necropsy performed, and tissues collected for laboratorytesting including quantitative gross lung lesion scoring,histopathology, IDEXX ELISA, virus titration, and virus neutralization.

TABLE 7 Time course for vaccination-challenge trial Day −28 Creation ofARP vaccines begins Day −21 Pigs are born on the source farm Day −7 Pigsarrive at ISU (3 weeks old), collect blood Day 0 First vaccination Day14 Collect blood Day 28 Collect blood, Second vaccination Day 42 Collectblood Day 49 Collect blood, challenge with virulent virus Day 49-70Monitor for clinical signs daily Day 56 Collect blood Day 63 Collectblood Day 70 Euthanize and necropsy pigs. Evaluate lesions, collecttissues for diagnostic testing and histopathology.

Pathology

At necropsy, gross pathology lung lesions were determined by a treatmentblinded, board certified veterinary pathologist as previously describedby Halbur et al. Halbur P G, Paul P S, Meng X J, Lum M A, Andrews J J,Rathje J A. Comparative pathogenicity of nine US porcine reproductiveand respiratory syndrome virus (PRRSV) isolates in a five-week-oldcesarean-derived, colostrum-deprived pig model. J Vet Diagn Invest 1996;8:11-20.

Briefly, each section of the lung was assigned a number and the lungscore represents the percentage of that lobe with evidence of pneumonia.The scores for each lobe are then added together to get the overall lungscore for that pig. The lung pathology in this experiment was rathermild and is summarized in FIG. 4 as the total lung score by group.Histopathology was also conducted on lung and heart sections asdescribed by Halbur et al., supra

These results are summarized in FIGS. 5-7. The interstitial pneumonia isthe average of four different sections per pig using a scale of 0(normal) to 6 (severe and diffuse). Results indicated no statisticaldifferences between groups. Lung lymphoid hyperplasia was examined onthe same sections with a scale of 0 (normal) to 6 (severe hyperplasia).Results indicated that both the placebo and inactivated vaccine groupshad statistically (p<0.05) greater hyperplasia when compared to thestrict negative group. No other statistical differences were noted. Theheart section was examined for signs of infection on a scale of 0(normal) to 3 (severe myocarditis). Results indicated no statisticaldifferences between groups.

The pathology results indicate few statistical differences despite thefact that other assays we conducted indicate a successful challenge.Lesions and clinical signs induced in PRRSV studies can vary greatly, orbe nearly absent as seen here, due to a variety of reasons includingstrain differences. This contributes to the fact that other measuressuch as virema and antibody assays discussed below are considered thegold standard for evaluating PRRSV vaccines.

IDEXX HerdChek PRRS 2XR ELISA

The IDEXX ELISA detects antibodies to the nucleopcapsid (N) protein ofPRRSV. These antibodies dominate the early response to PRRSV althoughthey do not provide protection from disease. Previous work has alsocorrelated the S/P (sample to positive, the ratio of sample signal tobackground that is commonly used with IDEXX ELISA) titers to level ofviremia and in this way the titers can be used to compare level ofinfection between groups. Johnson W, Roof M, Vaugh E,Christopher-Hennings J, Johnson C R, Murtaugh M. Pathogenic and humoralimmune response to porcine reproductive and respiratory syndrome virus(PRRSV) are related to viral load in acute infection. VeterinaryImmunology and Immunopathology 2004; 102:233-47.

Serum samples were collected from all pigs on 0, 7, 14, and 21 days postchallenge. Samples were sent to South Dakota State University fortesting. An S/P ratio of ≧0.40 is considered positive. The results areshown in FIG. 8 as mean S/P ratio per group. The results indicate thaton the day of challenge all pigs in Groups 1-4 remained negative. Thisassured us that we did not have lateral introduction of live virusduring the study. All pigs in Group 5 were IDEXX positive on the day ofchallenge which is to be expected since the N protein is a major part ofthe MLV vaccine. All pigs in the ARP group remained negative prior tochallenge reinforcing the idea that this vaccine can be used inconjunction with the IDEXX ELISA to differentiate infected fromvaccinated animals (DIVA). Groups 2-4 seroconverted by 14 days postchallenge. However the ARP group remained signicantly lower than theother treatment groups through necropsy indicating a lower level ofinfection in this group. This comparison was not possible in the MLVgroup since it was positive prior to challenge. The strict negativesremained negative throughout the study.

Fluorescent Focused Neutralization Assay

The fluorescent focused neutralization (FFN) assay detects serumneutralizing antibodies against PRRSV. Serum samples were collected fromall pigs on 0, 7, 14, and 21 days post challenge. Samples were sent toSouth Dakota State University for testing as previously described. Thetest virus used was PRRSV strain Pennway and an inverse titer ≧4 wasconsidered positive. The number of positive pigs in each group issummarized in FIG. 9. There no positives in any group on day ofchallenge. However after challenge the ARP and MLV groups showed morepigs positive by necropsy when compared to to the inactivated andplacebo groups. All pigs in the strict negative group remained negativethroughout the study. With more positive pigs it is not surprising thatthe mean neutralizing titers of the ARP and MLV groups were higher thanthe other treatments as shown in FIG. 10.

Although our previous work has indicated that expressed PRRSV GP5 and Mcan induce neutralizing antibody prior to challenge we did not see thatin this study. However it is possible that this is due to glycosylationof the GP5 used for this study. This was a new vaccine created from afield strain that tend to be highly glycosylated. Although there werenot neutralizing antibodies prior to challenge, we did note a primingeffect in the ARP group similar to the MLV group as evidenced by thedifferences between groups post challenge.

Live Virus Titration

Serum samples collect at 0, 7, 14, and 21 DPC as well as bronchialalveolar lavage (BAL) fluid collected at necropsy were tested for thepresence of live PRRSV as previously described. Briefly, samples werediluted 10-fold on 96 well plates containing confluent Marc-145 cells.Each clinical sample was plated in quadruplicate. Plates were incubatedat 37° C. and 5% CO₂ for 7 days, or until no new CPE was observed.TCID₅₀/ml titers were calculated using the Reed-Muench equation. Thedetection limit was 5.6e1 TCID₅₀/ml.

All groups were negative for live virus on 0 DPC and the strict negativegroup remained negative throughout the study. By 7 DPC virus wasdetected in the four challenged groups and significant differences inviremia were noted (FIG. 11). There was no detectable live virus in theserum at 14 and 21 DPC. Testing of the BAL fluid indicated only onepositive sample in Groups 2 and 5 and two positive samples in Groups 3and 4 (Data not shown).

RT-PCR

RT-PCR was conducted on serum samples collected at 0, 7, 14, and 21 DPCas previously described. Briefly, viral RNA was extracted using theQiagen Virus Spin Kit. The extract was then tested in duplicate usingprimers specific for the ORF7 gene of PRRSV which codes for the Nprotein. The results are summarized as the number of positive pigs ineach group (FIG. 12).

While PCR is another assay to detect viremia, it is not surprising thatthe PCR and live virus titration results do not match. One methoddetects live virus while the other only detects nucleic acid which couldpossibly be present in the absence of live virus. The PCR resultsindicate that by 14 DPC the ARP and MLV groups had significantly fewerviremic pigs compared to the placebo group.

Example 2

In this study, the alphavirus replicon is derived from the TC-83 strainof the alphavirus Venezuelan Equine Encephalitis Virus (VEEV). In aprevious study, a VEEV replicon vaccine expressing the HA gene from ahuman H5N1 isolate protected chickens from lethal challenge.Schultz-Chemy S, Dybing J K, Davis N L, Williamson C, Suarez D L,Johnston R, Perdue M L. Influenza virus (A/HK/156/97) hemagglutininexpressed by an alphavirus replicon system protects chickens againstlethal infection with Hong Kong-origin H5N1 viruses. Virology. 2000 Dec.5; 278(1):55-9. PubMed PMID: 11112481. Recently, our group became thefirst to evaluate VEEV replicon particle vaccines in swine. Erdman M M,Kamrud K I, Harris D L, and Smith J, 2010, “Alphavirus Replicon ParticleVaccines Developed for Use in Humans Induce High Levels of Antibodies toInfluenza Virus Hemagglutinin in Swine: Proof of concept”, Vaccine28:594-596. The objective of this study was to evaluatereplicon-expressed recombinant novel H1N1 HA protein as a swine vaccinein a vaccination-challenge model.

Materials and Methods Novel H1N1 HA Replicon Subunit Vaccine Production

The hemagglutinin (HA) nucleotide sequence was retrieved from the GlobalInitiative on Sharing Avian Influenza Data (GISAID) database. The genewas synthesized by a commercial company (DNA2.0, Menlo Park, Calif.,USA) with unique AscI and PacI restriction sites engineered at the 5′and 3′ ends, respectively. The HA gene was cloned into the AscI/PacIsites of the pVEK (TC-83) replicon vector (Hooper J W, Ferro A M, GoldenJ W, Silvera P, Dudek J, Alterson K, Custer M, Rivers B, Morris J, OwensG, Smith J F, and Kamrud K I, 2009, “Molecular Smallpox VaccineDelivered by Alphavirus Replicons Elicits Protective Immunity in Miceand Non-Human Primates”, Vaccine 13(13)). and an optimized construct wasselected as previously described. (Kamrud K I, Custer M, Dudek J M,Owens G, Alterson K D, Lee J S, Groebner J L, Smith J F. Alphavirusreplicon approach to promoterless analysis of IRES elements. Virology.2007 Apr. 10; 360(2):376-87. Epub 2006 Dec. 6. PubMed PMID: 17156813;PubMed Central PMCID: PMC1885372). The HA gene was then sequenced toensure the proper sequence was maintained through the cloning process.RNA transcripts were produced in vitro as previously described (Kamrudet al. (2007), supra. Replicon RNA was mixed with Vero cells inelectroporation cuvettes and pulsed. Cells were incubated overnight andthen lysed using RIPA buffer (Pierce, Rockford, Ill., USA). Resultinglysate was tested for protein expression by Western blot and HA proteinconcentration was determined by a novel H1N1 HA-specific ELISA. Lysatewas diluted to the specified HA concentration and vaccine was adjuvantedwith Emulsigen-D (MVP Technologies, Omaha, Nebr., USA).

Western Blot Analysis

Vero cell lysate containing recombinant HA protein was separated byrunning on a 12% SDS-PAGE gel (Invitrogen, Carlsbad, Calif., USA) andwas then transferred to a PVDF membrane (Invitrogen, Carlsbad, Calif.).The ladder used was the SeeBlue Plus2 Pre-Stained Standard (Invitrogen,Carlsbad, Calif., USA). After transfer, membrane was blocked with 5%non-fat dry milk at room temperature. Membrane was incubated with swinepolyclonal anti-HA for two hours, washed three times, followed byincubation with goat anti-swine IgG horseradish peroxidase conjugate(ImmunoJackson Research Laboratories, Inc, West Grove, Pa., USA) for onehour, and washed three times. Detection was performed using TMBsubstrate (KPL, Gaithersburg, Md., USA).

Animal Studies

Pigs free of swine influenza virus (SIV) and porcine reproductive andrespiratory syndrome virus (PRRSV) were obtained at three weeks of age.Pigs were randomized and separated into 4 groups of 5 pigs each. Priorto vaccination, serum was collected and tested by the homologoushemagglutination inhibition (HI) assay against the novel H1N1A/California/04/2009 strain to confirm negative antibody status. Serawere collected throughout the study and tested by this same HI assay tomonitor seroconversion post-vaccination. A prime/boost vaccinationschedule was followed. The first dose of vaccine was given to pigs atapproximately 4 weeks of age on day 0. On day 21 pigs received boostervaccination, with challenge on day 47 and necropsy on day 52. Pigsreceived either phosphate buffered saline (PBS) (Group 1) or differentconcentrations of novel H1 HA recombinant protein (Groups 2-4,). Pigswere challenged intratracheally with virulent A/California/04/2009(CDC#2009712047) at a dose of 2×10⁵ TCID₅₀/ml. Nasal swabs werecollected daily for live virus isolation beginning on day of challengeand continuing until study completion 5 days post-challenge. Pigs wereweighed immediately before challenge and again at necropsy fordetermination of average daily gain. At necropsy, gross lung lesionconsolidation was determined by a board-certified pathologist. Lungtissue was fixed in formalin for SIV immunohistochemistry (IHC) andhistopathological analysis. Bronchoalveolar lavage fluid (BALF) wascollected from lungs for live virus isolation. This animal study wasapproved by the Iowa State University Institutional Animal Care and UseCommittee.

Gross Lung Lesion Scoring, Histopathology, and SIV Immunohistochemistry

A single board-certified veterinary pathologist who was blinded to grouptreatments, performed gross lung scoring, histopathological analysis,and SIV Immunohistochemistry (IHC) analysis. At necropsy, each lung lobeaffected by pneumonia was visually estimated, and a total percentage forthe entire lung was calculated based on weighted proportions of eachlobe to the total lung volume (Halbur P G, Paul P S, Frey M L, LandgrafJ, Eernisse K, Meng X J, Lum M A, Andrews J J, Rathje J A. Comparison ofthe pathogenicity of two US porcine reproductive and respiratorysyndrome virus isolates with that of the Lelystad virus. Vet Pathol.1995 November; 32(6):648-60. PubMed PMID: 8592800). Tissue samples fromthe trachea and all lung lobes were collected and fixed in 10% formalin.Tissues were routinely processed and stained with hematoxylin and eosin.Lung samples were scored according to the method used by Vincent et al.(Vincent A L, Ma W, Lager K M, Janke B H, Webby R J, García-Sastre A,Richt J A. Efficacy of intranasal administration of a truncated NS1modified live influenza virus vaccine in swine. Vaccine. 2007 Nov. 19;25(47):7999-8009. Epub 2007 Sep. 29. PubMed PMID: 17933442; PubMedCentral PMCID: PMC2099695). Swine influenza virus IHC was done accordingto the method described by Vincent et al. (Vincent L L, Janke B H, PaulP S, Halbur P G. Demonstration of swine influenza virus informalin-fixed, paraffin-embedded tissues by use of animmunohistochemical test. Proceedings of the 14th IPVS Congress. 1996;97). All tissue preparation and staining was done by the Iowa StateUniversity Veterinary Diagnostic Laboratory.

Live Virus Isolation

Live virus titers were determined from nasal swabs and live virusisolation performed on bronchoalveolar lavage fluid (BALF) samples.Briefly, nasal swabs and BALF samples were thawed and centrifuged toremove cellular debris. The resulting supernatant was diluted 10-fold in96 well plates in Dulbecco's Modified Eagle Medium (DMEM) (Gibco,Carlsbad, Calif., USA) containing 1% antibiotic-antimycotic (Gibco,Carlsbad, Calif., USA) and 1% L-glutamine (Mediatech, Manassas, Va.,USA). After dilutions were made, 1000 was transferred from each wellinto respective wells of a 96 well plate which contained a monolayer ofswine testicle (ST) cells. Plates were incubated at 37° C. until nofurther CPE was observed, typically 3-5 days. Wells displaying CPE wereconsidered positive, and titers were calculated using the TCID₅₀/mlmethod of Reed-Meunch. (Reed L T and Muench H. A simple method ofestimation of 50% end points. American journal of Hygiene. 27; 493-497.1938)

Hemagluttination Inhibition Assay

Antibodies to influenza virus were measured by hemagglutinationinhibition (HI) assay run by the University of Minnesota VeterinaryDiagnostic Laboratory following standard laboratory protocol. Briefly,sera were treated with receptor-destroying enzyme, heat inactivated,adsorbed with 20% turkey erythrocytes, and centrifuged. Supernatantswere then serially diluted in V-shaped well microtiter plates with anequal volume containing 4-8 agglutinating units of A/California/04/2009and plates were incubated at room temperature before addition of 0.5%turkey erythrocytes. Titer was defined as the reciprocal of the maximaldilution at which hemagglutination was inhibited.

Direct Antigen Capture ELISA

Unknown samples, negative controls, and purified novel H1 protein(Protein Sciences, Meriden, Conn., USA) were directly captured to NUNCMaxisorp (Rochester, N.Y., USA) 96-well microplates by diluting withcapture buffer (50 mM Carbonate/Bicarbonate, pH 9.6) and incubatedovernight at 4° C. (100 μl/well). The microplates were washed four timeswith wash buffer (20 mM Phosphate Buffered Saline, 0.05% Tween-20, pH7.2). The plates were blocked with 1.25% non-fat dry milk in capturebuffer for 1 hour at 37° C. (200 μl/well). After four washes, pigpolyclonal anti-HA was added to wells (100 μl) and incubated for 1 hourat 37° C. (diluted 1/500 in wash buffer containing 1.25% NFDM).Following four washes, goat ant-pig IgG-HRP labeled (JacksonImmunoResearch, West Grove, Pa., USA) was added to the wells (100 μl)and incubated for 1 hour at 37° C. (diluted 1/2000 in was buffercontaining 1.25% NFDM). Four final washes were performed prior to theaddition of 100 μl of TMB substrate (KPL, Gaithersburg, Md., USA) andincubation at 37° C. for 20 minutes. Absorbance values were measured at620 nm and a standard curve was plotted with the purified novel H1protein. Linear regression analysis of the standard curve was used tocalculate the novel H1 concentrations in the unknowns.

Statistical Analysis

Single factor analysis of variance (ANOVA) was used to analyzehomologous HI titers, macroscopic and histopathological lung scores, IHCand BALF results, log 10 transformed nasal swab viral titers, and ADG(JMP 8.0.1, SAS Institute Inc., Cary, N.C., USA). Statisticalsignificance was set at p<0.05.

Results Vaccine Preparation

The novel H1N1 HA gene was inserted into the alphavirus repliconplatform according to the methods listed previously. Nucleotidesequencing after insertion confirmed the correct HA gene sequence hadbeen maintained throughout the cloning process. Western blottingperformed on protein lysate confirmed expression of the novel HA proteinat all the varying HA doses used in vaccine preparation for the animalstudy. The HA concentration was determined by novel HA ELISA and dilutedto the specified HA concentration (Table 8). See FIG. 13.

TABLE 8 Design of novel H1N1 recombinant HA vaccine study. Group VaccineHA concentration/dose 1 Sham NA 2 Recombinant HA 1.14 μg 3 RecombinantHA 0.57 μg 4 Recombinant HA 0.38 μg Pigs received either sham vaccine(PBS, Group 1) or varying doses of HA antigen (Groups 2-4). All vaccineswere given intramuscularly as 2 ml doses on days 0 and 21.

Antibody Titers

Post-vaccination sera were tested for specific antibody response by thehomologous HI assay. Hemagglutination inhibition titers were not seen invaccinated pigs after one dose, but were all positive (?1:40), exceptfor a single pig in Group 2, at 7 and 14 days post-boost vaccination(data not shown). On the day of challenge, homologous HI titers weresignificantly higher in Groups 2-4 than Group 1 (Table 9).

TABLE 9 Summary of serum antibody titers, average macroscopic andmicroscopic lung involvement, immunohistochemistry (IHC), and averagedaily gain (ADG). HI % Histopathologic Lung Group Titers^(a)Pneumonia^(b) Score^(c) IHC^(d) ADG^(e) 1 <10  15.6 ± 5.4 1.8 ± 0.1 5/51.76 ± 0.2 2 121*  1.4 ± 0.9* 0.8 ± 0.2* 1/5* 2.56 ± 0.68 3 184*  0.2 ±0.2* 0.6 ± 0.2* 0/5* 2.64 ± 0.22* 4 106*  1.8 ± 1.1* 0.8 ± 0.2* 1/5*2.45 ± 0.34* ^(a)Geometric mean homologous HI liters ^(b)Group mean ±standard error ^(c)0-3, group mean ± standard error ^(d)Number ofpositive samples per group ^(e)ADG post-challenge in pounds, group mean± standard error *Values are significantly different from non-vaccinates(Group 1) within a column at p < 0.05

Pathological Evaluation

At necropsy, lungs exhibited macroscopic dark purplish-red consolidatedlesions located mainly in the cranioventral lobes. Lungs taken fromGroups 2-4 exhibited significantly lower lesion scores and consolidationthan pigs in Group 1 (Table 3). There was also a significant reductionin pathological scores in all vaccinated groups compared to thenon-vaccinated group (Table 3). The lung sections taken fromnon-vaccinated Group 1 pigs had approximately 50% of the airwaysaffected by bronchiolar epithelial disruption and peribronchiolarlymphocytic cuffing. The vaccinated Groups 2-4 demonstrated onlyoccasional affected airways with light cuffing. Swine influenza virusIHC was also performed on lung sections. All 5 lungs taken fromnon-vaccinated Group 1 pigs were positive for influenza antigen, whileonly 2 pigs in total from the vaccinated Groups 2-4 were positive.Additionally, SIV IHC was done on trachea samples taken from each pig atnecropsy (data not shown). Although there were positive trachea IHCsamples in all groups, there was no significant differences betweenvaccinated and non-vaccinated groups. Positive trachea IHC resultscorrelate with what was previously reported on pathogenesis of novelH1N1 in ferrets. (Munster V J, de Wit E, van den Brand J M, Herfst S,Schrauwen E J, Bestebroer T M, van de Vijver D, Boucher C A, Koopmans M,Rimmelzwaan G F, Kuiken T, Osterhaus A D, Fouchier R A. Pathogenesis andtransmission of swine-origin 2009 A(H1N1) influenza virus in ferrets.Science. 2009 Jul. 24; 325(5939):481-3. Epub 2009 Jul. 2. PubMed PMID:19574348)

Virus Isolation

No live influenza virus was detected one day post-challenge from nasalswabs (Table 4). On day 2 post challenge live influenza virus wasdetected in Groups 1, 3, and 4, although there were no significantdifferences between mean group viral titers. On day 3 post-challengeGroups 2 and 4 had significantly lower titers than did Group 1. On bothdays 4 and 5 Groups 2-4 all exhibited lower titers than Group 1. No livevirus was detected in nasal swabs from any pigs in Group 2 for theduration of the challenge period. Similarly, there was a significantreduction in the number of positive BAL samples between groups (Table3). By 5 days post-challenge, only a total of 3 vaccinated pigs haddetectable live virus in BAL samples, while all 5 pigs in thenon-vaccinated group were virus isolation positive.

Average Daily Gain

All pigs were weighed on the day of challenge and again at necropsy.Groups 3 and 4 had significantly higher average daily gain (ADG) overthe 5 day period following challenge than did Group 1. Group 2 didexhibit higher ADG but was not significantly higher than Group 1(p=0.08).

TABLE 10 Summary of live virus isolation from nasal swabs andbronchoalveolar lavage (BAL). Nasal Swab^(a) BAL^(b) Group 1 DPC^(c) 2DPC 3 DPC 4 DPC 5 DPC 5 DPC 1 0 0.85 ± 0.53 2.55 ± 0.66 3.05 ± 0.18 3.05± 0.24 5/5 2 0 0 0* 0* 0* 2/5 3 0 1.05 ± 0.07 0.65 ± 0.65  0.9 ± 0.57* 1.0 ± 0.62* 0/5 4 0 0.45 ± 0.45  0.5 ± 0.5*  0.65 ± 0.65*  0.65 ± 0.65*1/5 ^(a)Log₁₀ mean virus titers ± standard error in nasal swabspost-challenge ^(b)Number of positive BAL samples per group ^(c)Dayspost-challenge (DPC) *Values are significantly different fromnon-vaccinates (Group 1) within a column at p < 0.05

A similar study was designed that did not involve an influenza viruschallenge or associated analysis; only immunogenicity of recombinant HAvaccines produced in this manner was measured in vaccinated animals. Forthis study the HA genes for three other influenza viruses (H1-beta,H1-delta, H1-gamma and H3) were inserted into the alphavirus repliconplatform using methods as described above. Nucleotide sequencing afterinsertion confirmed the correct HA gene sequences had been maintainedthroughout the cloning process. Western blots were performed on proteinlysates generated with each of the HA constructs to confirm expressionof the HA protein (data not shown). In this experiment varying dilutionsof the resultant HA vaccines were used to vaccinate groups of pigs(vaccines used and schedule described in the Tables 11 and 12 below);the dilutions used to vaccinate pigs are shown in Table 11. The immuneresponses induced by the different recombinant HA vaccines were analysedby homologous HI titers, using the method described above where endpointtiter is shown as the reciprocal of the last dilution of serum capableof inhibiting hemagglutination of the virus in the assay. A summary ofthe HI titers determined in this study can be found in Table 12.

TABLE 11 Day Task −7 Collect blood, treat w/antibiotics, tag, randomize0 Vaccinate pigs 21 Collect blood, treat with booster vaccine dose 28Collect blood for serology 35 Collect blood for serology 42 Collectblood, treat with 2^(nd) booster vaccine dose 57 Euthanize animals,collect large volume blood samples

TABLE 12 Vaccine schedule HI titer Dilution # of # of (μg./ml) Antigen(1 mL dose) doses animals 7 dpb FLUVENT-beta 1:60 3 2 FLUVENT-beta 1:753 3 FLUVENT-delta 1:60 3 2 FLUVENT-delta 1:75 3 3 FLUVENT-gamma 1:60 3 2FLUVENT-gamma 1:75 3 3 FLUVENT-H3 1:60 3 2 FLUVENT-H3 1:75 3 3 Noglyc +PwayM + ORF7 1:10 3 2 Noglyc + PwayM 1:10 3 2

TABLE 13 Hi titer 21 days post boost Vaccine:dilution reciprocal HItiter H1-beta 1:60 160 H1-beta 1:60 10 H1-beta 1:75 160 H1-beta 1:75 160H1-beta 1:75 320 H1-delta 1:60 160 H1-delta 1:60 80 H1-delta 1:75 80H1-delta 1:75 160 H1-delta 1:75 320 H1-gamma 1:60 320 H1-gamma 1:60 40H1-gamma 1:75 160 H1-gamma 1:75 80 H1-gamma 1:75 10 H3 1:60 320 H3 1:6040 H3 1:75 320 H3 1:75 160 H3 1:75 320

Discussion

The outbreak of novel H1N1 in the human population has highlighted thezoonotic potential that influenza viruses possess. Even before thepandemic of this decade, there were many reported cases of swine tohuman transmission of influenza. As such, part of controlling thiszoonotic threat is vaccination of swine against swine influenza viruses.In this study, we demonstrate how rapidly an efficacious swine influenzavaccine based on the alphavirus replicon expression system can beproduced in response to an outbreak of a novel zoonotic strain. Thisreports on immunization of swine with a recombinant protein produced viaan alphavirus replicon expression system. Replicon particle (RP)vaccines produced with this system have recently been utilized to induceprotection against swine influenza virus (SIV) and porcine reproductiveand respiratory syndrome virus (PRRSV) in swine. (Erdman M M, Kamrud KI, Harris D L, Smith J. 2010, Alphavirus Vector Vaccines Developed forUse in Humans Induce High Levels of Antibodies to Influenza VirusHemagglutinin in Swine: Proof of Concept. Vaccine 28:594-596; BosworthB, Erdman M, Stine D, Harris I, Irwin C, Jens M, Loynachan A, Owens G,Kamrud K, Harris D L. 2010, Virus-like replicon particle vaccineprotects pigs against influenza Comparative Immunology, Microbiology andInfectious Diseases 33 (2010) e99-e103. The first proof of concept studydemonstrated that a replicon particle vaccine administered to swine wasable to induce high antibody HI titers against a human influenza strain.A subsequent study using an RP vaccine expressing the HA gene of a GladeIV H3N2 SIV isolate confirmed that influenza HA RP vaccines given toswine are not only able to induce an antibody response, but also providesignificant protection against a homologous viral challenge. In contrastto these earlier studies, this study used an alphavirus repliconexpression system to produce recombinant HA protein in vitro; however,similar antibody response and protection from viral challenge wasdemonstrated.

The results demonstrate that influenza infection in swine withA/California/04/2009 is able to induce clinical symptoms and grosslesions comparable to other strains of SIV. (Vincent A L, Ma W, Lager KM, Janke B H, Webby R J, García-Sastre A, Richt J A. Efficacy ofintranasal administration of a truncated NS1 modified live influenzavirus vaccine in swine. Vaccine. 2007 Nov. 19; 25(47):7999-8009. Epub2007 Sep. 29. PubMed PMID: 17933442; PubMed Central PMCID: PMC2099695;Vincent A L, Lager K M, Ma W, Lekcharoensuk P, Gramer M R, Loiacono C,Richt J A. Evaluation of hemagglutinin subtype 1 swine influenza virusesfrom the United States. Vet Microbiol. 2006 Dec. 20; 118(3-4):212-22.Epub 2006 Aug. 1. PubMed PMID: 16962262; Sreta D, Kedkovid R, TuamsangS, Kitikoon P, Thanawongnuwech R. Pathogenesis of swine influenza virus(That isolates) in weanling pigs: an experimental trial. Virol J. 2009Mar. 25; 6:34. PubMed PMID: 19317918; PubMed Central PMCID: PMC2678088.)In contrast with a previous study, several pigs (primarily in thenon-vaccinated group) in this study exhibited clinical signs, mainlycoughing and sneezing. This discrepancy may be due to the miniature pigmodel used in the previous study (Itoh Y et al. In vitro and in vivocharacterization of new swine-origin H1N1 influenza viruses. Nature.2009 Aug. 20; 460(7258):1021-5. PubMed PMID: 19672242; PubMed CentralPMCID: PMC2748827). In this study, vaccine administration inducedspecific antibody titers, reduced macroscopic and histopathologic lunglesions, and reduced viral load in both the nose and lung. Vaccinatedpigs also demonstrated a higher average daily gain than non-vaccinates.These results demonstrate that this recombinant novel HA protein isefficacious when used as a vaccine against novel H1N1 swine influenza.

This study also demonstrated the quickness and flexibility with which avaccine can be produced using the alphavirus replicon expression system.It took less than two months from the time the novel HA sequence wasretrieved from GISAID database until pigs were administered the firstvaccine dose. Traditional methods for producing influenza vaccines takemuch longer and are dependent on viral replication in embryonated eggsor on tissue culture cells with subsequent inactivation. In the face ofan influenza epidemic, a quick turnaround is important in preventingfurther transmission and decreasing the zoonotic potential. Thealphavirus replicon platform allows for rapid insertion of any influenzaHA (or other) gene, making it an attractive influenza vaccine technologydue to the constant antigenic shift and drift among influenza viruses.Novel H1N1 has not been identified in pigs in the United States.However, it has been confirmed to exist in swine herds in severalcountries, including neighboring Canada. (ProMED-mail. InfluenzaPandemic (H1N1) 2009, Animal health (08): Singapore, Swine. ProMED-mail;04 Sept: 20090904.3114. www.promedmail.org. 2009; OIE World AnimalHealth Information Database. Vol 22 Nos. 18-40. April 30-Oct. 1, 2009.http://www.oie.int/wahis/public.php ?page=weekly_report_index&admin=0.2009.) Several recent studies have already reported the successfultransmission of novel H1N1 virus from infected to naïve contact pigs.(Brookes S M, Irvine R M, Nunez A, Clifford D, Essen S, Brown I H, VanReeth K, Kuntz-Simon G, Loeffen W, Foni E, Larsen L, Matrosovich M,Bublot M, Maldonado J, Beer M, Cattoli G. Influenza A (H1N1) infectionin pigs. Vet Rec. 2009 Jun. 13; 164(24):760-1. PubMed PMID: 19525527;Lange E, Kalthoff D, Blohm U, Teifke J P, Breithaupt A, Maresch C,Starick E, Fereidouni S, Hoffmann B, Mettenleiter T C, Beer M,Vahlenkamp T W. Pathogenesis and transmission of the novel swine-origininfluenza virus A/H1N1 after experimental infection of pigs. J GenVirol. 2009 September; 90(Pt 9):2119-23. Epub 2009 Jul. 10. PubMed PMID:19592456.) Therefore, vaccination of pigs against novel H1N1 may preventor delay the virus' introduction into the United States swine populationor decrease its' occurrence in both swine and human populations, and mayalso increase consumer confidence in pork products.

Tissue or fluids from animals at a location where pigs have been exposedto influenza virus is obtained. Using the RT-PCR approach as describedin Example 1, HA of the virus is isolated. The sequence encoding HA isintroduced into a vector and lysis of vero cells infected with thereplicon for production of HA antigen (and mixed with an appropriateadjuvant) or, in another experiment, an RP vaccine produced using theprocedures described in Example 1. Pigs are administered a vaccine andmorbidity and mortality results measured.

Example 3

Foot-and-mouth disease (FMD) is a highly infectious disease ofcloven-hoofed animals that rapidly spreads by contact and aerosol.(Bachrach HL. Foot-and-mouth disease: world-wide impact and controlmeasures. In: Kurstak E, Maramorosch K, editors. Viruses andenvironment. New York: Academic Press; 1978. p. 299-310.) Outbreaks ofFMD in Taiwan, Japan, South Korea, the United Kingdom, and theNetherlands, countries that had been FMD-free for many decades, resultedin significant adverse economic consequences including slaughter oflarge numbers of animals and loss of export markets. Even the verylimited FMD outbreak in the United Kingdom in the summer of 2007, whichresulted from the escape of FMDV from the Pirbright facility that housesboth the government Institute of Animal Health and the Merial vaccinemanufacturing laboratory, resulted in significant economic losses (˜$100million US). These outbreaks demonstrate the vulnerability of FMD-freecountries, such as the US, to this disease. Disease control proceduresinclude restriction of animal movement, slaughter of infected andexposed animals, and vaccination in certain situations. However, currentvaccines, which are chemically inactivated preparations of live virus,have a number of shortcomings including the inability to induce rapidprotection.

An RP vaccine that co-expresses the FMDV capsid and 3C proteinase codingregions was produced by engineering the capsid-3C proteinase cassetteinto a replicon vector and generating RP that could be used to immunizecattle. See GenBank Accession No. AY593768 (2005). The capsid-3Cproteinase cassette was obtained from officials at the USDA, ARS, ARS,NAA Plum Island Animal Disease Center Foreign Animal Disease ResearchUnit. Data from these vaccinations is shown. in the FIG. 19 and Table14.

TABLE 14 Lesion scoring 5 Days post-FMDV challenge Foot inspectedTreatment Left Right Right Left Animal ID group Tongue front front rearrear D10-27 T02 Pos Neg Neg Neg Neg D10-32 T02 Pos Neg Neg Neg NegD10-33 T02 Pos Neg Neg Pos Neg D10-35 T02 Pos Neg Neg Neg Neg D10-26 T03Neg Neg Neg Neg Neg D10-28 T03 Pos Pos Pos Pos Pos D10-30 T03 Pos NegNeg Neg Neg D10-31 T03 Pos Pos Pos Pos Pos D10-29 T01 Sham Pos Pos PosPos Pos D10-34 T01 Sham Pos Pos Pos Pos Pos

Cattle vaccinated one time with 1×10⁹ IU A24 RP showed nearly completeprotection from systemic disease after FMDV challenge (one animal showedlesions on one hoof while all others remained symptom free). Half of theanimals that received 5×10⁸ IU of A24 RP were protected from significantsystemic disease. Dose A: 1×10⁹ IU. Dose B: 5×10⁸ IU. Numbers shown overthe columns pre-challenge represent the # positive animals/total #animals. Virus neutralization assays were run on serum collected at 0,7, 14 and 21 days post vaccination. Results are shown below. All of theanimals in the highest dose group demonstrated FMDV neutralizingantibodies by day 7 that were maintained through the day of challenge(day 21).

Two candidate rapid response vaccine approaches against foot and mouthdisease virus (FMDV) are described here. The first consists of repliconRNAs that express FMDV genes packaged into particles (RP). A similar RPas described above is prepared using an autogenous source and animalsvaccinated as outlined below. The purified RP represent the vaccine. Thesecond consists of a protein lysate generated by introducing repliconRNAs that express FMDV genes into cells; a recombinant subunit (RS)lysates vaccine is then harvested by lysing the transfected cells aftervaccine antigen has been produced. Both replicon-based approachesprovide the ability to differentiate between vaccinated and infectedanimals.

Replicon vectors co-expressing the FMDV capsid and 3C proteinase codingregions of FMDV are prepared. Expression of these FMDV proteins producesvirus-like particles (VLP) because the 3C proteinase processes the coatproteins allowing them to self-assemble into antigenic VLP. The P1-2Acapsid coding region, the 2B coding region and the complete 3C proteasecoding region will be expressed. (Moraes M P, Mayr G A, Mason P W,Grubman M J. Early protection against homologous challenge after asingle dose of replication-defective human adenovirus type 5 expressingcapsid proteins of foot-and-mouth disease virus (FMDV) strain A24.Vaccine 2002; 20:1631-9.) Because most of the FMDV nonstructuralproteins are not included in the genes engineered into the repliconvector animals vaccinated with this can be unequivocally differentiatedfrom infected animals.

Replicon clones expressing at the highest relative level and whichproduct the highest titer RP are selected. Monoclonal antibodies areused that are cross reactive with all seven serotypes of FMCV in yieldand expression analysis.

The first approach is to generate an RNA subunit (RS) vaccine byintroducing the replicon RNA that expresses the FMDV capsid and 3Cproteinase coding regions into cells in culture. Once the repliconcarrying the FMDV genes has been introduced into cells each of theindividual cells express the FMDV proteins. The FMDV proteins expressedin the cells are harvested by lysing the cells creating an FMDV proteinlysate that constitutes the RS vaccine. In brief, RNA transcripts willbe produced in vitro (Promega RiboMAX transcription system) from thereplicon plasmid and purified by either spin-column (gel binding andelution) or size exclusion chromatography, followed by agarose gelanalysis to assess integrity, and quantification by ultraviolet (UV)absorbance. Specified mass amounts of the replicon RNA will be mixedwith certified Vero cells in electroporation chambers and pulsed usingoptimal conditions for transfection efficiency and protein expression.Electroporated cell suspensions will be seeded into individual rollerbottles with media containing serum and incubated at 37° C. in 5% CO₂for 18-24 hr. Following incubation, cells are trypsinized and pelletedby centrifugation. Cells are then lysed by resuspending the cell pelletwith a mammalian cell lysis buffer (RIPA Lysis and Extraction buffer,Thermo Scientific). The resultant lysate is tested for potency viaWestern blot analysis to confirm protein expression.

In RS potency assays, densitometry analysis of Western blots specificfor FMDV VP2 capsid protein will be used to determine a relativeconcentration of FMDV antigen. The relative antigen concentration willbe associated with a total cellular protein concentration determinedusing the BCA Protein Assay Reagent (bicinchoninic acid, Pierce,Rockford, Ill.) method and a bovine serum albumin protein standardcurve. The maximum concentration of FMDV antigen will be based on theminimum formulation dilution possible and volume restrictions linkedwith practical vaccination of the test animals. In addition to the mostconcentrated FMDV antigen dose, two additional dilutions of FMDV antigenlysates will be formulated. The two additional dilutions will representa 1:5 and a 1:10 dilution of the highest dose.

The second approach is to generate an FMDV RP vaccine. FMDV RP vaccinesare produced by introducing into Vero cells by electroporation areplicon RNA that expresses the FMDV genes and two helper RNAs. FMDV RPare then harvested from the cells approximately 18 hours postelectroporation; the RP express the FMDV capsid and 3C proteinase codingregions when introduced into animals by vaccination. In brief, RNAtranscripts will be produced in vitro as described above from thereplicon and helper plasmids and purified by either spin-column (gelbinding and elution) or size exclusion chromatography, followed byagarose gel analysis to assess integrity, and quantification by UVabsorbance. Specified mass amounts of the replicon and helper RNAs willbe mixed with certified Vero cells in electroporation chambers andpulsed using various electroporation parameters to identify the optimalconditions for transfection efficiency and RP yield. Electroporated cellsuspensions will be seeded into individual roller bottles containingserum-free medium and incubated at 37° C. in 5% CO₂ for 18-24 hr.Following incubation, media and cells from the roller bottles will becombined and pumped through a charged depth filter. RP will be elutedfrom the cells and filter using a high NaCl concentration buffer andstored at −80° C. until ready for use. The infectious titer of the RPpreparation will be measured by antigen-specific IFA and tested atdefined MOI in a CPE assay to assure the absence of detectablereplication-competent virus. After a negative result is obtained fromthe CPE assay, the RP preparation is considered devoid of detectable RCVand can subsequently be handled under BL 1 laboratory conditions.

Potency assays are used to determine RP titer based on an IFA assay andqRT-PCR analysis to determine the number of RNAs associated with eachRP. Determining the total number of RNA copies helps to assure vaccineconsistency from serial to serial. The method for calculating thepotency is based upon an IFA specific for the vaccine H3 antigen. The H3positive cells are observed and quantified. Individual wells of the IFAtissue culture plate are visualized under 10× magnification and wellscontaining 20 to 50 H3 positive cells per grid field are used. A totalof five fields per well are counted. A duplicate well is counted in thesame manner. An average of the ten readings is used to calculate thepotency, or RP/ml. The total number of H3 positive cells is determinedby inserting the average of the ten counts into the following equation:potency=(average)×(dilution)×(100)/(0.12) where average represents theaverage of ten positive H3 cell counts for the sample, dilutionrepresents the well in which the average H3 positive cells were counted,100 is a constant representing the surface area of the wells in thetissue culture plate and 0.12 is a constant representing the volume ofRNA particle vaccine tested (ml).

Densitometry analysis of Western blots specific for FMDV VP2 capsidprotein will be used to determine a relative concentration of FMDVantigen. The relative antigen concentration will be associated with atotal cellular protein concentration determined using the BCA ProteinAssay Reagent (bicinchoninic acid, Pierce, Rockford, Ill.) method and abovine serum albumin protein standard curve. The maximum concentrationof FMDV antigen will be based on the minimum formulation dilutionpossible and volume restrictions linked with practical vaccination ofthe test animals. In addition to the most concentrated FMDV antigendose, two additional dilutions of FMDV antigen lysates will beformulated. The two additional dilutions will represent a 1:5 and a 1:10dilution of the highest dose.

Animals

Species/Breed/Strain: Bovine, no restriction on breed or strainSex: No restrictionsApproximate Initial Age: 3-6 months at time of vaccination. No weightrestriction and/or weight (Day 0).Approximate Number: 10 enrolledSource of Supply/Origin: Animals sourced from commercial farm orproduction systemIdentification: Each animal will be identified by a uniquely numberedear tagConditioning/Acclimation: Acclimated ≧5 days prior to administration ofinvestigational veterinary product (IVP)Management/Housing: Animals will be fed and watered in accordance withthe standard procedures of the study site. Animals will be handled incompliance with site Institutional Animal Care and Use Committee (IACUC)approvals and site facility regulations.Exclusion: Only clinically healthy, animals will be enrolled in thestudy. Unsuitable animals will be excluded from the study at thediscretion of the Investigator and/or the attending veterinarian priorto the administration of the IVP. Reasons for any animal being removedfrom the study will be included in the final report.Allotment: The identification number of each enrolled animal will berecorded on the allocation plan prior to the administration of the IVP.

TABLE 15 Investigational Veterinary Product Generic ProductReplication-defective RP vectored Foot-and-Mouth Name Disease Virussubunit vaccine pERK-A24 RP (as in FIG. 3 with the PRRSV gene replacedwith the FMDV sequences) Formulation RP are formulated in 1.0% fetalbovine serum, 5% sucrose, 200 mM sodium chloride in 10 mM sodiumphosphate, pH 7.3. In Vitro Assay Sterility and titer; RCV Results TestArticle Unused material will be retained for potential use Retention inadditional studies depending on study outcome Applied Dose 1 dosecontaining 5 × 10⁸ - or 1 × 10⁹ IU/mL 2 mL per dose VaccinationIntramuscular (IM) Route

TABLE 16 Challenge Strain Foot-and-Mouth Disease Virus (FMDV) serotypeA24 Cruzerio, SGD strain Source DHS/PIADC experimentally passaged oncein bovine Storage ≦−70° C. IDL Challenge Approximately 1-2 × 10⁴ bovineinfectious dose Dose 50 (BID₅₀) per animal IDL Challenge Intradermalinoculation at multiple sites in the tongue/0.5 Route/Volume 1.0 totalmL. This route of challenge in cattle is one recommended by the OIE.

TABLE 17 Study Groups Route of Vaccine # of # of Admin- Dose Dose Doses/Treatment Vaccine Animals istration Volume IU Animal T01 Control 2 IM 2mL N.A. 1 (sham immunized) T02 pERK-A24 4 IM 2 mL 1 × 10⁹ 1 RP T03pERK-A24 4 IM 2 mL 5 × 10⁸ 1 RP

Vaccination and Challenge

On Day 0, blood from all cattle will be collected (baseline). Cattlewill be vaccinated once with test article (T02, T03) or sham-immunized(T01) at Day 0. On Day 7 and 14 blood from all cattle (T01-T03) will becollected and tested for the presence of serum virus neutralizing (SVN)antibodies to FMDV A24. Cattle will be challenged with FMDV serotype A24Cruzerio SGD strain according to OIE guidelines. For challengeadministration, each animal will be sedated and then receive intradermalinoculations at multiple sites (0.5-1.0 total mL) in the upper surfaceof the tongue.

An RP or RS vaccine is especially useful as there currently is noapproved FMDV vaccine in the US. Rather, the US would have to rely uponsources in other countries, and those vaccines would be unlikely to beeffective in US strains and biotypes. With the present invention, a USbased FMDV could be biotypes, and vaccine prepared quickly. Tissue orfluids from animals at a location where animals have been exposed toFMDV virus is obtained. Using the RT-PCR approach as described inExample 1, FMDV capsid and 3C proteinase coding regions of the virus areisolated. The sequences are introduced into a vector and lysis of verocells infected with the vector or, in another experiment, an RP vaccineproduced using the procedures described in Example 1. Animals areadministered a vaccine and morbidity and mortality results measured.

Example 4

The following demonstrates use of interfering RNA and autogenouslysourced as a vaccine for animals. Those experiments below in which IMNVvaccines and WSSV VP28 vaccines were prepared used a shrimp farm as thesource of the nucleic acid of interest, first amplified in disease freeanimals before sequencing.

Determination of RNAi Sequences

In order to evaluate candidate sequences that would induce RNAi inresponse to IMNV, in vitro dsRNA was synthesized corresponding toregions of viral genome. Template DNA for in vitro transcription wascreated by extracting viral RNA using a commercial nucleic acidpurification kit (Qiagen RNeasy Mini). cDNAs to IMNV genome were createdusing specific oligionucleotide primers designed from sequencesavailable (GenBank accession no. EF061744). (Senapin, S., Phewsaiya, K.,Briggs, M., Flegel, T. W., 2006. Outbreaks of infectious myonecrosisvirus (IMNV) in Indonesia confirmed by genome sequencing and use of analternative RT-PCR detection method. Aquaculture 266, 32-38.) Reversetranscription (Thermoscript RT Invitrogen) was performed by adding 5 uLof RNA extract to the reaction mix and incubated at 50 degrees for 60minutes per manufacturer's instructions. Following reversetranscription, template cDNA (−50 ng) was added to a PCR master mix(PuReTaq Ready-To-Go PCR Beads) and thermocycling was performed usingoligonucleotideprimers to specific regions of the IMNV genome (Table18). Cycling conditions were 95° C. for 4:00 followed by 35 cycles at94° C. for: 30, 55° C. for: 30, 72° C.1:00 and a final extension of10:00 at 72° C.

dsRNA sequences used in experiments 1-3 (see list of sequences at end):dsRNA#3 (SEQ ID NO: 1)dsRNA#3 5′ Truncate (SEQ ID NO: 2)dsRNA#3 3′Truncate (SEQ ID NO: 3)dsRNA #2 (SEQ ID NO: 4)dsRNA#1 (SEQ ID NO: 5)GFP dsRNA (SEQ ID NO: 6)

TABLE 18 Oligonucleotide Primer Sequences Primer Sequence 5′-3′ eGFPT7FTAATACGACTCACTATAGGGAGAATGGTGAGCAA (SEQ ID NO: 7) GGGCGAGGAGCTGT eGFPT7RTAATACGACTCACTATAGGGAGATTACTTGTACA (SEQ ID NO: 8) GCTCGTCCATGCCG Pep195FAGAAAGTTTGTTTCGTAGAGCGAGA (SEQ ID NO: 9) Pep1474RAAAGGTGGCAGGTGTCCATACTGA (SEQ ID NO: 10) Pep1 95 T7FTAATACGACTCACTATAGGGAGAAGAAAGTTTGT (SEQ ID NO: 11) TTCGTAGAGCPep1474 T7R TAATACGACTCACTATAGGGAGAAAAGGTGGCAG (SEQ ID NO: 12)GTGTCCATAC Capsid4 F AATTTGGGTGGTTGGGACACATGG (SEQ ID NO: 13) Capsid 4 RCCCGACTTTCGTGCACACAACTTT (SEQ ID NO: 14) Capsid4T7 FTAATACGACTCACTATAGGGAGAAATTTGGGTGG (SEQ ID NO: 15) TTGGGACACACapsid4 T7R TAATACGACTCACTATAGGGAGACCCGACTTTCG (SEQ ID NO: 16) TGCACACRdRP1 F TCAACTCACTCGCAGCTGAAGGTA (SEQ ID NO: 17) RdRP1 RAATATAGCAACGTCGTCTCCGCGT (SEQ ID NO: 18) RdRP1 T7 FTAATACGACTCACTATAGGGTCAACTCACTCGCA (SEQ ID NO: 19) GCTGAAG RdRP1 T7 RTAATACGACTCACTATAGGGAATATAGCAACGTC (SEQ ID NO: 20) GTCTCCG VP19 T7 FTAATACGACTCACTATAGGGAGACGAAGCTTGGC (SEQ ID NO: 21) CACCACGACT VP19 T7 RTAATACGACTCACTATAGGGAGACGGAGCTCCTG (SEQ ID NO: 22) CCTCCTCTTGGGGTAAVP28F CGGGATCCATTGAAGGCCGCGCCATGGATCTTTC (SEQ ID NO: 23) TTTCACTCT VP28RCGGAGCTCTTACTCGGTCTCAGTGCCAGA (SEQ ID NO: 24) VP28 AscI FGAGAGGCGCGCCATGGATCTTTCTTT (SEQ ID NO: 25) VP28 PacI RTCTCTTAATTAACTACTCGGTCTCAGT (SEQ ID NO: 26) AscPep 1 anti FCTAAGGCGCGCCTAAAGGTGGCAGG (SEQ ID NO: 27) -ssdsRNA#3CGCGTTAATTAAAGAAAGTTTGTTTCG (SEQ ID NO: 28)

Products were then cloned into pCR4.0 vectors (Zero Blunt TOPO PCRcloning kit, Invitrogen) and transformed into E. coli (TOP10,Invitrogen). Plasmids preparations from these transformants were used asthe template source for in vitro dsRNA synthesis. dsRNA was preparedusing Ambion MEGAscript® RNAi Kit following manufacturer's directions.Briefly, opposing T7 RNA polymerase can be used at 5′ ends of one DNAtemplate or a single T7 promoter at opposite ends of a region to betranscribed is used with two templates, or two templates transcribed tomake complementary RNA molecules that are annealed. DNA templates fortranscription were PCR products with addition of T7 promoter sequencesamplified using the primer sequences described. (See, e.g. Ujvari, A andMartin, Conn. Identification of a Minimal Binding Element within the T7RNA Polymerase Promoter. J. Mol. Biol. (1997) 273, 775-781; Sousa et al.(2003) “T7RNA polymerase” Prog Nucleic Acid Res Mol Biol 73:1-41.). PCRcycling conditions were 95° C. for 4:00 followed by 35 cycles at 94° C.for: 30, 61° C. for: 30, 72° C. 1:00 and a final extension of 10:00 at72° C. These clones were then incubated overnight (16 hours) at 37° C.forming dsRNA. dsRNA products were then incubated with DNase I and RNasefor 1 hour and purified using the provided columns. dsRNA synthesis wasconfirmed by gel electrophoresis in comparison with a molecular weightladder (pGEM ladder, Promega) and product was quantifiedspectrophotometrically (BioRad SmartSpec).

Animal Rearing

Specific pathogen free (SPF) postlarvae were received from ShrimpImprovement Systems (Plantation Key, Fla.) and reared in a biosecureanimal holding facility. Animals were placed into 1000 L Poly tankscontaining artificial seawater (Crystal Sea Marine Mix), an oystershellairlift biofilter, and an activated carbon filter. Animals were fed acommercial growout diet (Rangen 35/10, Buhl, Id.) until 5 grams inweight.

Preparation of Viral Inoculum

A modification of the methods from Hasson et al (Hasson, K. W.,Lightner, D. V., Poulos, B. T., Redman, R. M., White, B. L., Brock, J.A., Bonami, J. R., 1995. Taura syndrome in Penaeus vannamei:demonstration of a viral etiology. Disease of Aquatic Organisms 23,115-126) was used to make a clarification for viral inoculation.Briefly, whole frozen animals that tested positive for infection withIMNV by PCR were received from Shrimp Improvement Systems. Tail musclewas removed from these animals, diluted 1:3 in TN buffer (0.02 MTris-HCl, 0.4 M NaCl, pH 7.4) and homogenized in a sterilized Waringblender for 5 minutes. The macerate was placed into centrifuge tubes andcentrifuged at 4000×g. The supernatant was then removed and centrifugedagain at 15,000×g for 30 minutes. A final centrifugation step wasperformed at 25,000×g for 60 minutes. This supernatant was diluted 1:10in sterile 2% saline and filtered through 0.2 micron syringe filters(Whatman). This clarification was then aliquoted into cryotubes andfrozen at −80° C. for challenge studies.

Determination of Challenge Dose

A dilution series of inoculum was prepared by diluting the preparedstock virus 10 fold and 100 fold into sterile 2% saline. Stock virus, aten fold dilution, and a 100 fold dilution were injected into the thirdabdominal segment into groups of 20 naive SPF animals weighing 5-8 gramseach. Animals were observed twice daily for mortality. All dosesresulted in 100% mortality at varying time points. 100% mortality took 2days for stock concentration, 7 days for ten fold dilution, and 12 daysfor 100 fold dilution (FIG. 15). The 100 fold dilution of the stockvirus was used as the viral challenge dose for the described challengeexperiments.

Histopathology

Moribund animals that were found prior to death were fixed in Davidson'sfixative for 24 hours before being transferred to 70% EtOH. Tissues wereembedded in paraffin, cut into slides and stained with Hematoxylin andEosin and evaluated for the presence of IMNV lesions.

Inoculation of Animals

Double stranded RNA (dsRNA) was prepared by diluting dsRNA into RNasefree water to the specified concentration. 50 μL, was injected into themuscle of the third abdominal segment of animals using a tuberculinsyringe.

Study Design #1

Two hundred liter tanks containing synthetic seawater and an oystershellairlift biofilter were stocked with 20 SPF juveniles weighing 5-7 gramsand allowed to acclimate for 72 hours. Following acclimation, fourseparate dsRNA constructs corresponding to three different segments ofthe IMNV genome were evaluated by comparison to a hetereologous dsRNAcontrol. A total of 2 μg of in vitro synthesized dsRNA was inoculatedinto animals into randomized tanks. Following vaccination animals werechallenged 48 hours later with IMNV. Animals were counted daily formortality. Moribund animals were fixed for histopathology. Followingthis treatment, surviving animals from vaccination and challenge withdsRNA #3 were reared (n=16) for an additional 60 days and thenrechallenged with undiluted viral stock.

Study Design #2

Two hundred liter tanks containing synthetic seawater were stocked with10 animals weighing 5-7 grams that were allowed to acclimate for 72hours. Following acclimation, 6 experimental groups received a dose (2,0.2 or 0.02 μg) of a dsRNA construct (dsRNA #2 or dsRNA #3), controlgroups received 2 ug of a heterologous eGFP dsRNA control. These groupswere then split with one being challenged 2 days following vaccination,and another being challenged 10 days following vaccination. Mortalitywas assessed daily and moribund animals were fixed for histopathology.

Results Experiment #1

Two of the dsRNA constructs demonstrated protection against IMNVchallenge. dsRNA#2 (SEQ ID NO: 4) had 62% survival (38% mortality) incomparison to only 3% (97% mortality) for the non-vaccinated controls at30 days post vaccination. The effect of dsRNA #3 (SEQ ID NO: 1) was evenmore robust with over 80% survival (FIG. 16). Significant differences(P<0.05) were calculated between dsRNA #2 and #3-injected animals ascompared to controls according to Oneway ANOVA followed by Tukey'smultiple comparison test using SPSS software. No significant differenceswere evident between animals injected with dsRNA #1, dsRNA GFP or the 2%saline control. Non-challenge controls remained at 100% survivalthroughout the duration of the study. Animals receiving non-sequencespecific dsRNA had similar mortalities to the non-vaccinate group.

The surviving animals from dsRNA#3 (SEQ ID NO: 1) group had 100% (16/16)survival following the second challenge (60 days after the primary viruschallenge) with one hundred fold higher virus concentration. Thisindicates that protection from challenge is robust even after anextended period of time has passed between the first and second viralchallenge.

Experiment #2

The construct dsRNA #3 (SEQ ID NO: 1) showed excellent protection evenat the lowest dose and longest interval between vaccination andchallenge at 80% survival. (FIG. 18) In comparison, low doses of dsRNA#2(SEQ ID NO: 4) appeared to have little impact on survival with survivalrates of 10% and 30% following challenge. However, non-vaccinated groupshad no survival in either group and non-sequence specific administeredgroup had 10% survival when challenged at 48 hours (FIG. 17).Non-challenged controls had survivals of 100%. After 30 days,significant differences (P<0.05) were noted with dsRNA #2 and#3-injected animals as compared to controls according to Oneway ANOVAfollowed by Tukey's multiple comparison test using SPSS software. Nosignificant differences were evident between animals injected with dsRNA#1, dsRNA GFP or the 2% saline control.

Experiment #3

To determine whether the entire dsRNA#3 sequence was required to providethe protection noted above two additional dsRNAs that are simpletruncations of the full length dsRNA#3 were tested (dsRNA#3 5′ truncateand dsRNA#3 3′ truncate). Animals weighing 5-7 grams were used asdescribed above to test the new dsRNA#3 sequences (2 μg of each dsRNAwere used). The construct dsRNA #3 3′ truncate (SEQ ID NO: 3) was shownto be 100% protective when delivered 10 days post vaccination at a doseof 0.02 μg (FIG. 19). dsRNA 5′ truncate (SEQ ID NO: 2) and the originaldsRNA3 (SEQ ID NO: 1) were shown to be 90% protective at 25 days (15days post challenge) when challenged 10 days following vaccination.

These experiments show preferred sequences having a very strong effecton dose and duration of RNAi trigger components that will be deliveredthrough the described following vector systems.

Further truncations also showed protection, using the same proceduresdescribed above. A 153 bp sequence (dsRNA#3 223-376, SEQ ID NO: 61)showed 100% protection, a 81 bp sequence (dsRNA#3 194-275, SEQ ID NO:56) showed 100% protection and a 54 bp sequence (dsRNA#3 219-273, SEQ IDNO: 51) 73% protection.

Replicon Particles

Replicon particles were produced as described, supra.

Alphavirus Replicon Proof of Concept

Alphavirus replicon particles expressing structural proteins andantisense sequences of WSSV and IMNV virus were created by cloning genesfrom a commercially synthesized gene sequence (GeneArt) into an existingalphavirus backbone vector. The WSSV genes of interest, in this caseVP19, VP28, and the complementary sequence to VP19 (VP19 antisense) werecreated using a sequence derived from a virulent WSSV isolate fromThailand (AF369029.2 GI:58866698). In the case of IMNV sequences werederived from available sequence from an Indonesian isolate downloadedfrom GenBank (EF061744.1 GI:124303516), and reverse complement of theregion spanning from 95-474 bases of the published sequence. Geneconstructs were designed to include appropriate restriction sites onboth the 5′ and 3′ ends to facilitate cloning onto the replicon plasmid.Following insertion of the protective molecule into the replicon vector,the insert was sequenced to confirm the identity of the construct (IowaState University DNA Sequencing Facility). The replicon plasmid DNA waslinearized and used to generate RNA transcripts by run-off transcriptionusing a commercially available in vitro transcription kit (RiboMAX T7Express, Promega). Replicon RNA containing the target gene (VP28, VP19,VP19 antisense, IMNV-ssRNA of dsRNA#3), and the two helper RNAs thatcode for the VEE capsid or glycoprotein genes were prepared using thesame run-off transcription method. Specified (previously optimized) massamounts of the replicon and helper RNAs were mixed with Vero cells inelectroporation chambers and pulsed using previously optimized squarewave electroporation parameters. Electroporated cell suspensions wereseeded into roller bottles containing serum-free medium and incubated at37° C. in 5% CO2 for 16-18 hours. Replicon particles (RP) were harvestedfrom culture fluids and the infectious titer of the RP preparation wasmeasured by antigen-specific IFA and tested in a cytopathic effect (CPE)assay to assure the absence of detectable replication-competent virus.RP was purified by size exclusion/ionic exchange filtration. The potency(infectious titer) of the purified bulk RP will be determined by IFA andthe preparation will be formulated and frozen at −80° C.

Sequences insertions used in replicon plasmids (see list of sequences atend):

VP28 (SEQ ID NO: 29)

VP19 (SEQ ID NO: 30) encoding the protein, and also transcribed toproduce dsRNAVP19-antisense (SEQ ID NO: 31)VP19-IR (inverted repeat DNA producing dsRNA) (SEQ ID NO: 32)DNA transcribed to produce antisense strand of dsRNA#3) (SEQ ID NO: 33)

Red Florescent Protein (RFP) (SEQ ID NO: 34)

The titer (IU/ml) of the RP preparations generated was measured. Arepresentative example of the IU/ml titer of the RP preparationsgenerated is: VP-19 RP=1.4E8 IU/mL, VP-28 RP=1.2E8 IU/mL, and VP19-AntiRP=5.86E7 IU/mL, IMNV RNA3 antisense=3.49E8/ml IU/mL The RP preparationsall passed the CPE assay by demonstrating the absence of detectablereplication competent virus. Following release assays the RP wereconsidered appropriate for use in the studies described below. Forexperiment #5 an additional group of antisense VP19 replicons werecreated that allowed for an increase titer of 1.26E81 U/mL.

Preparation of WSSV for use as a challenge stock virus were amplifiedutilizing SPF stocks of L. vannamei with a virulent strain of WSSV (DonLightner, University of Arizona). Fifty (50) SPF juvenile shrimpweighing approximately 12 grams were exposed per os with infected tissuefrom moribund shrimp that were PCR positive for WSSV (infected tissuesused were stored at −80° C. prior to use). Infected tissues were diluted1:3 in TN buffer (0.02 M Tris-HCl, 0.4 M NaCl, pH 7.4) and homogenizedin a sterilized Waring blender for 5 minutes. The macerate was placedinto centrifuge tubes and centrifuged at 4000×g. The supernatant wasthen removed and centrifuged again at 15,000×g for 30 minutes. A finalcentrifugation step was performed at 25,000×g for 60 minutes. Thissupernatant was diluted 1:10 in sterile 2% saline and filtered through0.2 micron syringe filters (Whatman). This clarification was thenaliquoted into cryotubes and frozen at −80° C. for challenge studies.Challenge dose was then optimized by injecting SPF animals with serialdilutions of viral inoculum in 2% saline. A challenge dose of 1 partinoculum and 10,000 parts 2% saline resulted in 10-20% survival 14 daysafter challenge and was used for the viral challenge dose for thedescribed studies.

Viral Challenge: Experimental animals were challenged by injection 3days after the initial injection with RP. Experimental and controlgroups will be observed for mortality over a 21 day period. At thetermination of the experiment the remaining individuals were sedated andeuthanized in an ice slurry, fixed whole in Davidson's fixative, andsubmitted for histological analysis. A gill tissue sample was taken andfrozen at −20° C. for PCR testing if needed.

Experiment #4 Experimental Design

SPF juvenile L. vannamei weighing approximately 5 grams were placed into16 tanks containing 10 animals and synthetic seawater (Crystal SeaMarine Mix) at 30 ppt salinity and maintained at 25° C. Three replicatetanks were provided for each experimental group. Experimental animalswere injected with 50 uL of RP expressing VP28 (SEQ ID NO: 28) or VP19(SEQ ID NO: 30) at a concentration of 10E8 IU/mL or VP19 antisense RP(SEQ ID NO: 31) at a concentration of 10E7 IU/mL into the ventral sinus.A sham injection control group was injected with 50 μL of cell culturemedia used as a control. VP19 naked double stranded RNA (SEQ ID NOs: 49and 50) was used as a positive vaccine control as it had provided 100%protection in previous experiments.

Experiment #5 Experimental Design

SPF juvenile L. vannamei weighing approximately 5 grams were placed into15 tanks containing 10 animals and synthetic seawater (Crystal SeaMarine Mix) at 30 ppt salinity and maintained at 25° C. Experimentalanimals were injected with 50 uL of RP expressing VP19 at aconcentration of 10E8 IU/mL or VP19 antisense RP at a concentration of10E7 IU/mL into the ventral sinus. A sham injection control group wasinjected with 50 uL of cell culture media used as a control. VP19 nakeddouble stranded RNA was used as a positive vaccine control of 10 dayspost vaccination as it had provided 100% protection in previousexperiments

Experiment #6 Experimental Design

SPF larvae and postlarvae were be placed into petri dishes containing 25mL seawater and 10E7 IU/mL of RP expressing the RFP reporter protein.Immersion exposure was done at room temperature for 2 hours. Animalswere then transferred to 500 mL flasks containing seawater and anairstone. Whole larvae were sacrificed at 24, 48, and 72 hourspost-exposure and evaluated for fluorescence using epifluorescencemicroscopy. Control animals were immersed into tanks containing cellculture media, and evaluated using the same method. This study was usedto determine if a) RP infectivity remains intact through the digestivetract and b) RP are able to infect and express a foreign protein inlarval and post larval animals.

Experiment #7

SPF juvenile L. vannamei weighing approximately 5 grams were placed intotanks containing 10 animals and synthetic seawater (Crystal Sea MarineMix) at 30 ppt salinity and maintained at 25° C. To evaluate theduration of immune response for extended periods of time in animalsadministered specific dsRNA or non-specific dsRNA animals were givenspecific dsRNA (VP19 or VP28) or non-specific (eGFP) by IM injection (5μg) and challenged 3 days following administration. Following theprimary challenge in which only slight mortality was observed, a secondchallenge was performed 21 days later.

Experiment #8

In order to compare methods of delivery, and determine if an orallydelivered sequence was protective, SPF juvenile L. vannamei weighingapproximately 5 grams were placed into tanks containing 10 animals andsynthetic seawater (Crystal Sea Marine Mix) at 30 ppt salinity andmaintained at 25° C. Experimental animals were fasted for 24 hours,injected with 5 μg of dsRNA VP19 or reverse gavaged (enema) with 5 ugVP19 dsRNA diluted in sterile water. Animals were challenged 14 daysafter vaccine administration.

Results Experiment #4

At 21 days post challenge, VP19 dsRNA (Control), VP19 RP, VP19 antisenseRNA-RP or VP28 RP showed 100%, 70%, 40% and 40% survival respectively.The positive control group showed 20% survival (FIG. 20). This studydemonstrates that VP19 dsRNA and VP19 expressed by RP provide protectionagainst mortality due to WSSV. As seen in Experiment 1 and 7, protectionup to at least 24 days was observed. Referring to FIGS. 16, 17, 18 and19B. protection up to at least 30 and at least 40 days is provided. Thisstudy will be repeated and duration of this protection followinginoculation will be assessed.

Experiment #5

For the group that was challenged 3 days post vaccination, VP19 RP, VP19antisense —RP and positive control group showed 5%, 35% and 0% survival,respectively 14 days post challenge. For the group that was challenged10 days post vaccination VP19 dsRNA (Control), VP19 RP and VP19antisense RNA-RP showed 95%, 5% and 60% survival 21 days post challenge,respectively. The positive control group for the 10 day post vaccinationgroup showed 20% overall survival. See FIGS. 21 and 22.

Experiment #6

Fluorescence was difficult to evaluate in the post larval stages due toautofluorescence present in the gut tissue in both controls andexperimental groups. In contrast, the larval stages evaluated (Mysis andZoea) demonstrated strong specific RFP fluorescence in both gut andgills when compared with controls at 48 and 72 hours post inoculationwith RFP RP. This shows that protein can be delivered to the aquaticinvertebrate digestive tract and that immersion vaccination of larvalanimals provides a feasible delivery system for replicon particles.

Experiment #7

Differences in survival were observed between VP19 (100% survival), VP28(83%), Non-specific dsRNA (33.3%) and unvaccinated control (20%) (FIGS.23, 24) following challenge at 21 days. This demonstrates thedifferences between specific and non-specific dsRNA in the duration ofthe protective response.

Experiment #8

Animals administered VP19 dsRNA via IM and reverse gavage demonstratedprotection (95% and 100% survival respectively) versus controls. (FIG.25)

Experiment #9

Study Design: 200 L tanks were stocked with 3-5 gram SPF growth lineanimals and allowed to acclimate for 24 hours. Tanks were equipped withan oystershell airlift biofilter that has been allowed to mature in anLT tank with ammonia. Animals were divided into three groups, onereceiving dsRNA3 82 by dsRNA fragment, one receiving eGFP (greenfluorescent protein (GFP) gene (Sheen et al., Plant J. (1995)8(5):777-84) as a heterologous dsRNA control treatment, and onereceiving sterile water as a no dsRNA treatment. The dsRNA treatmentgroups received a 100 uL injection containing 5 μg of in vitrosynthesized dsRNA 2 days following challenge with IMNV. Animals werechallenged 2 days prior to dsRNA vaccination with a 1:100 dilution ofIMNV clarification. Groups were counted daily for 30 days and evaluatedfor mortality. Moribund animals were fixed in Davidson's solution forhistopathology and muscle tissues taken for qPCR analysis. Animals werefrozen at −80 degrees at termination of study.

Primers used for producing the dsRNA #3 were SEQ ID NO: 59, SEQ ID NO:60, to produce SEQ ID NO: 56. Primers for producing GFP included SEQ IDNO: 7, SEQ ID NO: 8 and SEQ ID NO: 6 is the DNA producing GFP dsRNA forcontrol.

TABLE 19 Number Challenge Treatments of shrimp Replications intervaldsRNA #3 2 dpc 10 3 −2 days dsRNA #3 2 dpc 10 −2 days dsRNA #3 2 dpc 10−2 days eGFP 2 dpc 10 3 −2 days eGFP 2 dpc 10 −2 days eGFP 2 dpc 10 −2days Challenge Control 10 3 −2 days Challenge Control 10 −2 daysChallenge Control 10 −2 days

Results

Animals receiving a treatment of dsRNA3 81 by demonstrated a 50%survival following challenge with IMNV. In comparison, animals receivingeither eGFP dsRNA or sterile water as a control demonstrated 0% survival(FIG. 26).

Conclusions

dsRNA 3 81 by can successfully reduce mortality when administered 2 dayspost infection with IMNV.

Experiment #9 Experimental Animals

Specific pathogen free (SPF) juvenile L. vannamei weighing 3-5 gramswere stocked into 200 L tanks (10 animals/tank) and allowed to acclimatefor 48 hours. Each tank contained artificial seawater with oystershellbiofilter and activated carbon.

Feed formulation

Chitosan encapsulated particles were prepared using VP19 dsRNA, supraSEQ ID NO: 30)., or IMNV dsRNA3 (not truncated, SEQ ID NO: 1). 0.2 gramschitosan was dissolved in 100 ml sodium acetate buffer. 1.0 mL of thissolution was then transferred to a new bottle with 99 ml of 50 mM sodiumacetate buffer (1:100 dilution), resulting in a 0.002% w/v solution ofchitosan. 120 ug of each dsRNA (VP 19 and dsRNA3) were diluted in asodium sulfate solution (0.2 M sodium acetate and 0.2 M acetic acid) toa total volume of 300 ul. 300 ul dsRNA solution was combined with 300 ul0.002% chitosan solution. The solution was heated in a 55 C water bathfor 1 minute, and promptly vortexed for 30 seconds. The tubes were thencentrifuged at 13,200×g for 10 minutes. Following the centrifugation,the solution was resuspended by pipetting and top coated onto 1 gramground feed. The entire 600 ul of the chitosan-dsRNA solution was addedfirst, followed by 600 ul 2% agarose. The feed was then blended withpipette tip to create evenly mixed clumps, which solidified afterseveral minutes.

Experimental Design

TABLE 20 Replications Survival Treatments # Shrimp (# of tanks) (%) 1.VP19 dsRNA 10 3 33% chitosan nanoparticles 2. IMNV dsRNA3 10 3 0chitosan nanoparticles 3. VP19 dsRNA 10 3 67% without chitosan 4.Positive control 10 3 0

After 3 days shrimp in each treatment group were challenged 100 uL with0.2 micron filtered WSSV clarification diluted in 2% sterile saline atWSSV 1:1×10⁵. Daily feeding of 10% biomass and 10% water exchange wasperformed daily to remove molts, excess food and fecal material.Mortality was observed for 21 days and samples of dead animals werefrozen at −80 for further testing.

Results

Following challenge with WSSV, animals that were treated with feedcoated with VP19 dsRNA demonstrated 67% survival. In addition, groupsthat were treated with feed coated with chitosan nanoparticlescontaining VP19 dsRNA demonstrated 33% survival following challenge withWSSV. Animals that received sham treatment in the feed (PositiveControl) had 0% survival following challenge. (See FIG. 27).

Experiment #10

HV156: dsRNA duration

The experiment demonstrated dsRNA81(194-275) and dsRNA3 (95-475)vaccination efficacy at 30 days post vaccination 15×200 L tanks werestocked with 3-5 gram SPF growth line animals and allowed to acclimatefor 24 hours. Tanks were equipped with an oystershell airlift biofilterthat has been allowed to mature in an LT tank with ammonia. The dsRNAtreatment groups received a 100 μL injection containing 2.0 μg of dsRNA(DE3 fermentation production lots see Timmons et al 2001) diluted inRNase free water. Animals were then challenged 30 days after dsRNAadministration with a lethal dose of IMNV via injection. Groups wereevaluated daily for 21 days following challenge and evaluated forclinical signs and mortality.

TABLE 21 Number Random of Repli- Challenge Challenge Tank Treatmentsshrimp cations Material interval Numbers DE3 dsRNA3 10 1 1:100 30 days LDE3 dsRNA3 10 1 1:100 30 days K DE3 dsRNA3 10 1 1:100 30 days E DE3dsRNA81 10 1 1:100 30 days C DE3 dsRNA81 10 1 1:100 30 days P DE3dsRNA81 10 1 1:100 30 days S Challenge Control 10 1 1:100 30 days QChallenge Control 10 1 1:100 30 days H Challenge Control 10 1 1:100 30days B

Results

The study was terminated on Day 51 (21 days post challenge). Upontermination survival in treatment groups was significantly higher(P<0.0001 using Tukey's HSD following One-Way ANOVA) than controls.Animals administered dsRNA3 had 100% survival following administrationof dsRNA3, animals given dsRNA81 had a mean survival of 93.33% (90%,90%, and 100%). Sham administration controls had 6.67% mean survival attermination (0%, 10%, and 10%) (FIG. 28).

Conclusions

dsRNA3 and dsRNA81 production lots made in DE3 E. coli are highlyprotective against lethal IMNV challenge up to 30 days postadministration.

1. A method of protecting an aquatic invertebrate animal from disease,the method comprising, a) identifying a target nucleic acid molecule ofa microorganism causing said disease; b) preparing a protective moleculeselected from the group consisting of: (i) a nucleic acid moleculeproducing a dsRNA wherein said dsRNA comprises a sequence that thatcorresponds to all or a portion of said target nucleic acid molecule ofsaid microorganism; (ii) a dsRNA molecule comprising at least one RNAmolecule that is capable of hybridizing to said target nucleic acidmolecule of said microorganism; (iii) an antisense RNA moleculecomprising a sequence that is the complement of said target nucleic acidmolecule of said microorganism; (iv) a nucleic acid molecule producingsaid antisense RNA; and (v) a replicon particle comprising any of themolecules of (i),-(iv); and c) administering said protective molecule toan aquatic invertebrate animal wherein said animal is protected fromsaid disease.
 2. The method of claim 1, wherein said at least one RNAmolecule comprising a sequence that corresponds to all or a portion ofsaid target nucleic acid molecule is selected from the group consistingof an RNA molecule of at least 10 bp, at least 20 bp, at least 30 by andat least 50 bp.
 3. The method of claim 1, wherein said dsRNA furthercomprises at least one RNA molecule that is the complement of all or aportion of said target nucleic acid molecule.
 4. The method of claim 3,wherein said at least one RNA molecule comprising a sequence thatcorresponds to all or a portion of said target nucleic acid molecule andsaid at least one RNA molecule that is the complement of all or aportion of said target nucleic acid molecule is selected from the groupconsisting of an RNA molecule of at least 10 bp, at least 20 bp, atleast 30 by and at least 50 bp.
 5. The method of claim 1, wherein saidanimal is shrimp.
 6. The method of claim 1, wherein said disease isselected from the group consisting of White Spot Syndrome Virus (WSSV)and Infectious Myonecrosis Virus (IMNV).
 7. The method of claim 1,wherein said target nucleic acid molecule is selected from the groupconsisting of a nucleic acid molecule encoding White Spot Syndrome Virus(WSSV) VP19 or a fragment thereof and WSSV VP28 or a fragment thereof.8. The method of claim 7, wherein said target nucleic acid molecule isselected from the group consisting of SEQ ID NO: 29 or a fragmentthereof and SEQ ID NO: 30 or a fragment thereof.
 9. The method of claim1, wherein said dsRNA is produced by a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 56, and SEQ IDNO: 61 and a fragment thereof wherein said molecules which produces aprotective response.
 10. The method of claim 3, wherein said RNA that isthat corresponds to all or a portion of said target nucleic acidmolecule is selected from the group consisting of SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 57, andSEQ ID NO: 62 and a fragment thereof, and said RNA that is thecomplement of all or a portion of said target nucleic acid molecule isselected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 53, SEQ ID NO: 58, and SEQ ID NO:63 and a fragment thereof.
 11. The method of claim 3, wherein said RNAthat that corresponds to all or a portion of said target nucleic acid isselected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 47, SEQID NO: 49 and fragments thereof, and said RNA that is the complement ofall or a portion of said target nucleic acid molecule is selected fromthe group consisting of SEQ ID NO: 48, SEQ ID NO: 50 and fragmentsthereof.
 12. The method of claim 1, wherein protective molecule isadministered such that said particle is delivered to the digestive tractof said animal.
 13. The method of claim 1, wherein said protectivemolecule is a replicon particle producing a dsRNA comprising at leastone RNA molecule capable of hybridizing to said target nucleic acidmolecule and at least one RNA molecule that is the complement of all ora portion of said target nucleic acid molecule and whereby saidprotective molecule is administered by a method selected from the groupconsisting of immersion of said animal in a solution comprising saidparticle and oral administration of said particle.
 14. The method ofclaim 13 wherein said target molecule selected from the group consistingof White Spot Syndrome Virus (WSSV) VP19 or fragment thereof, WSSV VP28or a fragment thereof and IMNV or a fragment thereof.
 15. The method ofclaim 1, wherein said protective molecule is a replicon particleproducing an antisense RNA molecule that is the complement of a targetnucleic acid molecule of IMNV.
 16. A method of protecting shrimp fromdisease, said method comprising administering to said shrimp a nucleicacid molecule selected from the group consisting a nucleic acid moleculeencoding a polypeptide of White Spot Syndrome Virus (WSSV) VP19 orfragment thereof and WSSV VP28 or a fragment thereof, wherein saidnucleic acid molecule, when administered to said shrimp, protects saidshrimp from said disease.
 17. The method of claim 16, wherein said WSSVVP 19 encoding nucleic acid molecule comprises SEQ ID NO: 30 or afragment thereof.
 18. The method of claim 16, wherein said WSSV VP 28encoding nucleic acid molecule comprises SEQ ID NO: 29 or a fragmentthereof.
 19. The method of claim 16, where said nucleic acid molecule isintroduced into an alphavirus particle and said alphavirus particle isadministered to said shrimp.
 20. The method of claim 1, wherein saidanimal is protected from said disease for at lest 20 days followingexposure to said disease.
 21. A method of producing a composition forprotecting an aquatic invertebrate animal from disease, comprising, a)identifying a target nucleic acid molecule of a microorganism causingsaid disease; b) preparing a protective molecule selected from the groupconsisting of: (i) a nucleic acid molecule producing a dsRNA whereinsaid dsRNA comprises a sequence that that corresponds to all or aportion of said target nucleic acid molecule of said microorganism; (ii)a dsRNA molecule comprising at least one RNA molecule that is capable ofhybridizing to said target nucleic acid molecule of said microorganism;(iii) an antisense RNA molecule comprising a sequence that is thecomplement of said target nucleic acid molecule of said microorganism;(iv) a nucleic acid molecule producing said antisense RNA; and (v) areplicon particle comprising any of the molecules of (i),-(iv); and c)preparing a composition comprising said protective molecule.
 22. Acomposition for administration to an aquatic invertebrate animal,comprising a protective molecule selected from the group consisting of:a) a nucleic acid molecule encoding a polypeptide of a microorganismwhich causes disease in an aquatic invertebrate animal; b) a nucleicacid molecule producing a dsRNA wherein said dsRNA comprises a sequencethat that corresponds to all or a portion of a nucleic acid molecule ofa microorganism that causes disease in said animal; c) a dsRNA moleculecomprising at least one RNA molecule that is capable of hybridizing to anucleic acid molecule of a microorganism that causes disease in saidanimal; d) an antisense RNA molecule comprising a sequence that is thecomplement of a target nucleic acid molecule of a microorganism whichcauses disease in said animal; e) a DNA molecule producing saidantisense RNA; and f) a replicon particle comprising any of themolecules of a), b), c), d), or e); wherein said protective molecule,when administered to an aquatic invertebrate animal, protects saidanimal from said disease.
 23. An isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 51, SEQ ID NO: 56, SEQ ID NO: 61and a fragment thereof which, when administered to an aquaticinvertebrate animal, protects said animal from a disease selected fromthe group consisting of White Spot Syndrome Virus (WSSV) and InfectiousMyonecrosis Virus (IMNV).
 24. An isolated nucleic acid molecule selectedfrom the group consisting of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 57, SEQ ID NO: 62, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 53, SEQID NO: 58, SEQ ID NO: 63 and a fragment thereof which, when administeredto an aquatic invertebrate animal, protects said animal from a diseaseselected from the group consisting of White Spot Syndrome Virus (WSSV)and Infectious Myonecrosis Virus (IMNV).